Salutaridinol 7-O-acetyltransferase and derivatives thereof

ABSTRACT

This invention provides a protein comprising consecutive amino acids, the amino acid sequence of which is illustrated in FIG.  8  (SEQ. ID No.14) or, a fragment thereof having at least 15 amino acids, or a variant thereof, the sequence of which has at least 70% identity with the amino acid sequence of FIG.  8  (SEQ. ID No.14) over a length of at least 400 amino acids. This invention further provides a peptide comprising a fragment of salutaridinol 7-O-acetyltransferase protein of at least 6 consecutive amino acids which is not present in other plant acetyltransferases as illustrated in FIG.  2  (SEQ. IDs No.8 to 12). This invention further provides a nucleic acid molecule comprising consecutive nucleotides (i) the nucleic acid sequence of which is illustrated in FIG.  9  (SEQ. ID No.13) or FIG.  10  (SEQ. ID No.15), or (ii) a fragment thereof having a length of at least 45 nucleotides, or (iii) a variant thereof, the sequence of which has at least 70% identity with the sequence of FIG.  9  (SEQ. ID No.13) or FIG.  10  (SEQ. ID No.15) over a length of at least 1200 bases, or a sequence complementary to (i), (ii) or (iii), or the RNA equivalent of any of (i), (ii), or (iii). This invention further provides a nucleic acid molecule comprising a fragment of a salutaridinol 7-O-acetyltransferase gene, at least 18 consecutive nucleotides unique to the salutaridinol 7-O-acetyltransferase gene and being chosen from the 5′ or 3′ untranslated regions of the sequence illustrated in FIG.  9  (SEQ. ID No.13), or a sequence which is complementary thereto. This invention further provides a method for producing pentacyclic morphinan alkaloids comprising (i) introducing a nucleic acid molecule encoding salutaridinol 7-O-acetyltransferase into a plant cell capable of expressing salutaridinol and/or salutaridine and/or (R)-reticuline, (ii) propagating said plant cell to produce a multiplicity of morphinan-producing cells, and (iii) recovering said morphinan(s) from said multiplicity of cells. This invention further provides a method for the production of thebaine comprising the steps of (i) contacting in vitro a protein having salutaridinol 7-O-acetyltransferase activity with salutaridinol and acetyl co-enzyme A at pH 8 to 9, and (ii) recovering the thebaine thus produced.

[0001] The present invention relates to proteins having salutaridinol7-O-acetyltransferase activity and to derivatives and analogs of theseproteins. The invention also relates to nucleic acid molecules encodingthe proteins, derivatives and analogs, and to their use in theproduction of plants having altered alkaloid profiles.

[0002] The opium poppy Papaver somniferum produces some of the mostwidely used medicinal alkaloids. The narcotic analgesic morphine and theantitussive and narcotic analgesic codeine are the most importantphysiologically active alkaloids from this plant. Nineteen totalsyntheses of morphine have been reported through 1999 (1). The mostefficient synthesis of morphine proceeded on medium scale with anoverall yield of 29% (2). Despite many years of excellent syntheticorganic chemistry concentrated on morphinans, a commercially feasibletotal chemical synthesis has not yet been achieved for morphine orcodeine.

[0003] The enzymatic synthesis of morphine in P. somniferum has beenalmost completely elucidated by M. H. Zenk and co-workers and issummarized by Kutchan (3). Morphine is derived from two molecules of theamino acid L-tyrosine in a series of at least seventeen enzymatic steps.The latter steps in the pathway that lead specifically from(S)-reticuline, a central intermediate of isoquinoline alkaloidbiosynthesis, to morphine involve three NADPH-dependent oxidoreductases(4-6), most probably three cytochromes P-450 (7) and an acetylCoA-dependent acetyltransferase (8).

[0004] Acetyl CoA-dependent acetyltransferases have an important role inplant alkaloid metabolism. They are involved in the synthesis ofmonoterpenoid indole alkaloids in medicinal plant species such asRauwolfia serpentina. In this plant the enzyme vinorine synthasetransfers an acetyl group from acetyl CoA to 16-epi-vellosimine to formvinorine. This acetyl transfer is accompanied by a concomitant skeletalrearrangement from the sarpagan- to the ajmalan-type (9). An acetylCoA-dependent acetyltransferase also participates in vindolinebiosynthesis in Catharanthus roseus, the source of the chemotherapeuticdimeric indole alkaloid vinblastine (10,11). AcetylCoA:deacetylvindoline 4-O-acetyltransferase catalyzes the last step invindoline biosynthesis.

[0005] Central to morphine biosynthesis in P. somniferum is acetylCoA:salutaridinol 7-O-acetyltransferase [EC 2.3.1.150] (FIG. 1).Acetylation of the phenanthrene salutaridinol is followed by allylicsyn-displacement of the acetylated (activated) hydroxyl by the phenolichydroxyl, which follows stereocontrol for S^(N)240 substitution ofcyclohexene rings, thereby producing the pentacyclic morphinan ringsystem (8).

[0006] Each of the known enzymes of morphine biosynthesis has beendetected in both P. somniferum plants and cell suspension culture, yetplant cell cultures have never been shown to accumulate morphine orcodeine (3). Morphine accumulation in the plant appears to be related todifferentiation of a latex system (12). Efforts aimed at the metabolicengineering of the P. somniferum alkaloid profile as well as atdeveloping alternate biotechnological sources of morphinans, have todate been hampered by lack of knowledge regarding suitable genetictargets. Indeed, only one gene specific to the morphine biosynthesispathway has been isolated and characterized to date (13).

[0007] The present invention provides and characterises both at the DNAand protein level, such a genetic target, namely salutaridinol7-O-acetyltransferase (SalAT) of morphine biosynthesis in P. somniferum.Derivatives and variants of the protein are also provided.

[0008] More specifically, the present invention relates to a proteincomprising or consisting of:

[0009] i) the amino acid sequence illustrated in FIG. 8 (SEQ. ID No. 14)or,

[0010] ii) a fragment of the amino acid sequence illustrated in FIG. 8(SEQ. ID No. 14), said fragment having at least 10 and preferably atleast 15 amino acids, or

[0011] iii) a variant of the amino acid sequence of FIG. 8 (SEQ. ID No.14), said variant having at least 70% identity with the amino acidsequence of FIG. 8 (SEQ. ID No. 14) over a length of at least 400 aminoacids.

[0012] A first preferred embodiment of the invention thus comprises thefull length salutaridinol 7-O-acetyltransferase protein whose amino acidsequence is shown in FIG. 8 (SalAT 1) (SEQ. ID No. 14). The protein ofthe invention as illustrated in FIG. 8 has 474 amino acids, and amolecular weight of approximately 52.6 kDa (Genebank accessionNo.AAK73661). According to this embodiment of the invention, the fulllength P. somniferum enzyme may be obtained by isolation andpurification to homogeneity from cell suspension culture, or from plantparts of P. somniferum, at any stage of development, and from latex ofmature or immature plants. Alternatively, the enzyme may be produced byrecombinant means in suitable host cells such as plant cells or insectcells. The protein may consist exclusively of those amino acids shown inFIG. 8 (SEQ. ID No. 14), or may have supplementary amino acids at the N-or C-terminus. For example, tags facilitating purification may be added.The protein may also be fused at the N- or C-terminus to a heterologousprotein.

[0013] The protein whose sequence is illustrated in FIG. 8 (SEQ. ID No.14) has salutaridinol 7-O-acetyltransferase activity.

[0014] In the context of the present invention, “salutaridinol7-O-acetyltransferase activity” signifies the capacity of a protein toacetylate 7(S)-salutaridinol at the C7 position to givesalutaridinol-7-O-acetate. This latter compound undergoes spontaneousallylic elimination at pH 8-9, leading to the formation of thebaine. AtpH 7, the allylic elimination leads to dibenz[d,f]azonine alkaloidscontaining a nine-membered ring. Salutaridinol 7-O-acetyltransferaseactivity is assayed according to Lenz and zenk (8). Specifically, anenzyme solution is combined with salutaridinol and acetyl coenzyme A.Enzyme activity is determined, either by decrease of salutaridinol, orby production of thebaine at pH 8-9.

[0015] According to a second embodiment of the invention, the proteinmay comprise or consist of a fragment of the amino acid sequenceillustrated in FIG. 8 (SEQ. ID No. 14), wherein said fragment has alength of at least 10 amino acids, preferably at least 12, or at least15 or at least 20 amino acids. By protein “fragment” is meant anysegment of the full length sequence of FIG. 8 (SEQ. ID No. 14) which isshorter than the full length sequence. The fragment may be a C- orN-terminal fragment having for example approximately 10 or 15 or 20amino acids, or may be an internal fragment having 10 to 40 amino acids.Preferably the protein fragments have a length of 15 to 470 amino acids,for example 20 to 450 amino acids, or 25 to 400 amino acids.Particularly preferred are fragments having a length of between 350 and450 amino acids, such as the FIG. 8 (SEQ. ID No. 14) sequence havingundergone truncation at the C- or N-terminal, or short peptides having alength of 10 to 25 amino acids, for example 15 to 23 amino acids.

[0016] The protein fragments of the invention may or may not havesalutaridinol 7-O-acetyltransferase activity. Normally, fragmentscomprising at least 400, or at least 450 consecutive amino acids of theprotein shown in FIG. 8 (SEQ. ID No. 14) are enzymatically active.

[0017] A particularly preferred class of peptides according to theinvention are peptides which comprise or consist of a stretch (or“tract”) of at least 5 or 6 amino acids unique to the salutaridinol7-O-acetyltransferase protein (SalAT) illustrated in FIG. 8 (SEQ. ID No.14). By “unique to SalAT” is meant a tract of amino acids which is notpresent in other plant acetyltransferases as illustrated in FIG. 2 (SEQ.ID Nos. 8 to 12). These SalAT-specific peptides typically have a lengthof 8 to 100 amino acids, for example 10 to 50 amino acids, or 15 to 20amino acids. Such peptides can be used for generation of SalAT-specificantibodies for immunodetection and immunopurification techniques.Examples of such short peptides are shown as white boxes in FIG. 2.

[0018] In general, the fragments may consist exclusively of part of theFIG. 8 (SEQ. ID No. 14) sequence. Alternatively, they may additionallycomprise supplementary amino acids which are heterologous to theillustrated P. somniferum enzyme, for example N- and/or C-terminalextensions. Such supplementary amino acids may be amino acids fromsalutaridinol 7-O-acetyltransferase enzymes from species other than P.somniferum, thus providing a chimeric salutaridinol7-O-acetyltransferase enzyme, or may be purification tags, fusionproteins etc.

[0019] According to a third preferred embodiment of the invention, theprotein comprises or consists of a variant of the amino acid sequence ofFIG. 8 (SEQ. ID No. 14). By “variant” is meant a protein having at least70% identity, and preferably at least 80% or 85% identity with the aminoacid sequence of FIG. 8 over a length of at least 400 amino acids.Particularly preferred are variants having at least 90% or at least 95%identity, for example 95.5 to 99.9% identity. Preferred variants havesequences which differ from the amino acid sequence illustrated in FIG.8 (SEQ. ID No. 14) by insertion, replacement and/or deletion of at leastone amino acid, for example insertion, replacement and/or deletion ofone to 10 amino acids, or one to five amino acids. Variants differingfrom the FIG. 8 (SEQ. ID No. 14) sequence by one to ten amino acidreplacements are particularly preferred, for example two, three, four orfive amino acid substitutions. Such variants may or may not havesalutaridinol 7-O-acetyltransferase activity, as defined previously.Preferably, the variants have this activity.

[0020] Particularly preferred “variant” proteins of the invention areallelic variants of SalAT, or SalAT proteins arising from expression ofother members of a SalAT gene family. The inventors have demonstratedthat within a given species of Papaver there exist variants of the SalATgene containing a number of single point polymorphisms, some of whichgive rise to changes in amino acid sequence. Typically, these variantscontain one to fifteen amino acid substitutions, for example one to ten,or one to six, with respect to the FIG. 8 (SEQ. ID No. 14) sequence.Amino acid changes are usually conservative, with a neutral amino acidsuch as isoleucine or serine being replaced by another neutral aminoacid such as valine or alanine, or an acidic amino acid such as asparticacid being replaced by another acidic amino acid such as glutamic acidetc. SalAT activity is usually conserved. An example of an allelicvariant is the enzyme shown in FIG. 11 (SEQ. ID No. 17), having fiveamino acid differences with respect to the FIG. 8 (SEQ. ID No. 14)sequence. The protein illustrated in FIG. 8 (SEQ. ID No. 14) will bereferred to as SalAT 1, and the variant shown in FIG. 11 (SEQ. ID No.17) as SalAT 2.

[0021] The protein variants of the P. somniferum. These variants, whichagain have at least 70% identity with the amino acid sequence of FIG. 8(SEQ. ID No. 14) over a length of at least 400 amino acids, preferablycontain the conserved amino acids shown as black boxes in FIG. 2 (SEQ.ID Nos. 7 to 12). Indeed, the amino acid sequence of the P. somniferumenzyme is similar to acyltransferases involved in monoterpenoid indolealkaloid-, phenylpropanoid conjugate- and diterpenoid formation (22,26-29). Histidine and aspartate residues (H₁₆₃-XXX-D₁₆₇) are highlyconserved as well as a DFGWG motif near the carboxy terminus of theproteins. The invention thus also includes variants of the FIG. 8 (SEQ.ID No. 14) protein having the required degree of identity with the FIG.8 protein (at least 70%) and including the DFGWG motif and the(H₁₆₃-XXX-D₁₆₇) motif. The equivalent histidine residue has been shownthrough site directed mutagenesis or chemical modification to beessential for catalytic activity in other acyltransferases (30).Carbethoxylation of histidine residues in salutaridinol7-O-acetyltransferase with DEPC resulted in a loss of enzyme activity.Preincubation of the enzyme with acetyl CoA partially protected aputative active site histidine residue from chemical modification andresultant inactivation. A catalytic triad (Ser-His-Asp) as found inserine proteases and lipases has been postulated for otheracyltransferases (30). The crystal structure of arylamineN-acetyltransferase from Salmonella typhimurium indicates that acysteine residue may be a component of the catalytic triad (C₆₉, H₁₀₇,D₁₂₂) (31). The amino acid sequence of salutaridinol7-O-acetyltransferase contains both a conserved serine (S₃₃) and aconserved cysteine (C₁₅₂) suggesting that a catalytic triad could alsobe essential to enzyme activity in this family of plantacyltransferases. This consensus information assists in theidentification and isolation of additional members of this family thatmay be involved in other plant secondary pathways.

[0022] The enzymatically active proteins of the invention, whether theyare variants or fragments as defined above, or the native P. somniferumenzyme shown in FIG. 8 (SEQ. ID No. 14), can be used for the in vitroproduction of alkaloids, particularly five-ringed morphinan alkaloids,such as thebaine. For example, according to the invention, thebaine canbe produced by:

[0023] i) contacting a protein of the invention having salutaridinol7-O-acetyltransferase activity with salutaridinol and acetyl co-enzyme Ain vitro at pH 8 to 9, and

[0024] ii) recovering the thebaine thus produced.

[0025] The SalAT proteins used in this in vitro method are generallyused in purified form.

[0026] In addition to the proteins described above, the invention alsorelates to nucleic acid molecule encoding such proteins, for examplecDNA, RNA, genomic DNA, synthetic DNA.

[0027] Examples of particularly preferred nucleic acid molecules aremolecules comprising or consisting of:

[0028] i) the nucleic acid sequence illustrated in FIG. 9 (SEQ. ID No.13) or FIG. 10 (SEQ. ID No. 15), or

[0029] ii) a fragment of the nucleic acid sequence illustrated in FIG. 9(SEQ. ID No. 13) or FIG. 10 (SEQ. ID No. 15), said fragment having alength of at least 18 nucleotides, preferably at least 30 nucleotides,and most preferably at least 45 nucleotides, or

[0030] iii) a variant of the sequence illustrated in FIG. 9 (SEQ.

[0031] ID No. 13) or FIG. 10 (SEQ. ID No. 15), said variant having atleast 70% identity with the sequence of FIG. 9 (SEQ. ID No. 13) or 10(SEQ. ID No. 15), over a length of at least 1200 bases, or

[0032] iv) a sequence complementary to sequences (i), (ii) or (iii), or

[0033] v) the RNA equivalent of any of sequences (i), (ii), (iii) or(iv).

[0034] The nucleic acid molecules (i), (ii), (iii), (iv) and (v) arealso referred to herein collectively as “the acetyltranferase gene orderivatives thereof”.

[0035] The nucleic acid molecule illustrated in FIG. 10 (SEQ. ID No. 15)is the coding region of the full length cDNA of P. somniferumsalutaridinol 7-O-acetyltransferase (Genebank accession No.AF339913).The invention encompasses any nucleic acid molecule which consistsexclusively of this sequence, or which additionally includes furthernucleotides at either the 5′ and/or 3′ extremities, for example, thesequence shown in FIG. 9 (SEQ. ID No. 13), which includes 5′ and 3′untranslated regions. The additional nucleotides may be otheruntranslated regions, or endogenous or exogenous regulatory sequences,or fusions to other coding regions.

[0036] Also within the scope of the invention are molecules comprisingor consisting of fragments of the nucleic acid sequence illustrated inFIG. 10 (SEQ. ID No. 15), said fragments having a length of at least 18nucleotides, preferably 30 nucleotides, and most preferably at least 45nucleotides, for example at least 60 or at least 90 nucleotides. In thecontext of the invention, a nucleic acid “fragment” signifies anysegment of the full length sequence of FIG. 10 (SEQ. ID No. 15) which isshorter than the full length sequence.

[0037] The fragment may be a 5′- or 3′-terminal truncation for example afragment of approximately 30 to 60 nucleotides, or an internal fragment.Preferred fragments have a length of 30 to 1400 nucleotides, for example50 to 1200 or 70 to 1000 nucleotides. Shorter fragments having a lengthof 18 or 30 to 150 nucleotides can be used as primers in nucleic acidamplification reactions, enabling the isolation of relatedacetyltransferases of species other than P. somniferum, or of differentlines within a given species of Papaver. When the nucleic acid fragmentof the invention is relatively short, i.e. between approximately 18 to50 nucleotides, it usually comprises a stretch (or tract) of at least 18nucleotides which is unique to the SalAT gene. Such unique tracts mayfor example encode protein fragments which do not occur in other plantacetyltransferases as shown in FIG. 2 (SEQ. ID Nos. 8 to 12), or may bechosen from untranslated regions. These fragments, or theircomplementary sequences, are useful in amplification reactions.

[0038] A preferred example of such SalAT-specific fragments arefragments which comprise or consist of a tract of at least 18 or 20consecutive nucleotides chosen from the 5′ or 3′ untranslated regions ofthe sequence illustrated in FIG. 9 (SEQ. ID No. 13), or a sequence whichis complementary thereto.

[0039] The longer nucleic acid fragments of the invention, which have alength of about 1200 to 1400 nucleotides, generally code for proteinswhich are enzymatically active and can therefore be used in the samemanner as the full length cDNA, for example in transformation of plantcells for production of alkaloids in vivo or in culture.

[0040] Molecules comprising fragments of the FIG. 10 (SEQ. ID No. 15)sequence also include genomic DNA which may contain at least one intron,and which can thus be considered to be an assembly of fragments linkedby one or more intronic sequences. Such a genomic molecule may furthercomprise the endogenous SalAT regulatory sequences.

[0041] The nucleic acid molecules of the invention may also be variantsof the sequence illustrated in FIG. 10 (SEQ. ID No. 15), said variantshaving at least 70% identity, and preferably at least 80%, at least 90%or at least 95% identity with the sequence of FIG. 10 (SEQ. ID No. 15),over a length of at least 1200 bases. Particularly preferred variantsshow 95 to 99.9% identity for example 96 to 99.5% identity. Mostpreferred variants differ from the sequence of FIG. 10 (SEQ. ID No. 15)by insertion, replacement and/or deletion of at least one nucleotide,for example replacement of one to two hundred nucleotides, or insertionof a total of 2 or more nucleotides, for example an insertion of 3 to100 nucleotides, whilst conserving at least 70% identity with the FIG.10 (SEQ. ID No. 15) sequence. An example of a sequence variant is asequence that is degenerate with respect to the sequence illustrated inFIG. 10 (SEQ. ID No. 15).

[0042] Typically, nucleic acid variants of the invention have thecapacity to hybridise to the sequence illustrated in FIG. 10 (SEQ. IDNo.15) in stringent conditions. Stringent conditions are for examplethose set out in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., USA,1989 pages 387-389, paragraph 11.

[0043] Particularly preferred nucleic acid variants of the invention arevariants of the SalAT gene occurring within a given species of alkaloidpoppy, such as allelic variants or gene family members. Allelic variantsusually have upto 1% difference in nucleotide sequence with respect tothe full length coding sequence, for example with respect to thenucleotide sequence shown in FIG. 10 (SEQ. ID No. 15), and usually sharethe same chromosomal location. The sequence of FIG. 11 (SEQ. ID No. 17)(SAT 2 CO48) is thought to be such a variant, arising from apolymorphism of the SalAT gene. Members of a gene family usually differby upto 5% with respect to the reference sequence and need not share thesame chromosomal location. The nucleic acid variants according to thisaspect of the invention are characterised in that they comprise at leastone nucleic acid substitution with respect to the FIG. 10 (SEQ. ID No.15) sequence, for example 2 to 30 base changes. The changes are usuallysingle base changes and may be silent or may give rise to amino aciddifferences.

[0044] The different polymorphic forms of the SalAT gene, such asalleles or gene family members, can be identified using amplificationtechniques with primers derived from the SalAT 1 sequence, particularlyprimers permitting amplification of the full coding sequence. Suitableprimers include portions of the reading frame, for example sequenceshaving a length of around 20 to 40 nucleotides and corresponding to the5′ and 3′ extremities of the coding sequence, for example immediatelydownstream of the ATG start codon and immediately upstream of the Stopcodon. Alternatively, other suitable primers correspond to parts of the5′ and 3′ untranslated regions, as illustrated in the Examples below.For example, RT PCR on mRNA from an alkaloid poppy, particularlyP.somniferum, can be carried out using a primer pair corresponding to astretch of around 30 bases upstream of the ATG start codon anddownstream of the stop codon in FIG. 9 (SEQ. ID No.13). These techniquespermit the identification of variants of the gene within a species, forexample P. somniferum. The primers may be adapted to allow inclusion ofa restriction enzyme site on the end of the amplification product tofacilitate cloning. The variants which can be identified and clonedusing such techniques are within the scope of the present invention.

[0045] Nucleic acid variants and fragments of the invention may encodean enzymatically active protein or not. Preferrred variants encodeproteins having salutaridinol 7-O-acetyltransferase activity, as definedpreviously.

[0046] The invention also encompasses nucleic acid molecules that arecomplementary to any of the foregoing molecules, variants and fragments.In the context of the invention, “complementary” means that Watson-Crickbase-pairs can form between a majority of bases in the complementarysequence and the reference sequence. Preferably, the complementarity is100%, but one or two mismatches in a stretch of twenty or thirty basescan be tolerated. Additionally, complementary stretches may be separatedby non-complementary stretches. Particularly preferred examples ofcomplementary sequences are antisense oligonucleotides and ribozymes,which can be used in alkaloid-producing plants such as poppies todown-regulate the production of salutaridinol 7-O-acetyltransferase, orrelated enzymes, thereby modifying the alkaloid profile of the plant.

[0047] The nucleic acid molecules of the invention can be used totransform or transfect eukaryotic and prokaryotic cells. To this end,the sequences are usually operably linked to transcription regulatorysequences such as promoters, transcription terminators, enhancers etc.The operable link between the acetyltransferase-derived coding sequenceand the regulatory sequence(s) may be direct or indirect, i.e. with orwithout intervening sequences, such as internal ribosome entry sites(IRES). The regulatory sequences may be endogenous to the codingsequence, i.e. they are the regulatory sequences naturally associatedwith the acetyltransferase sequence in the genome of the plant.Alternatively, the regulatory sequences may be heterologous to theacetyltransferase sequence. In this latter case the resulting constructforms a chimeric gene, comprising a coding sequence derived from theacetyltransferase gene, operably linked to at least one heterologoustranscription regulatory sequence. In the context of the invention, theterm “coding sequence” signifies a DNA sequence that encodes afunctional RNA molecule. The RNA molecule may be untranslated, or mayencode an enzymatically-active protein, or enzymatically-inactiveprotein. Particularly preferred promoters for plant expression areconstitutive promoters such as the 35S promoter, or tissue specific, ordevelopmentally specific promoters, or inducible promoters, dependingupon which expression pattern is sought.

[0048] The inventors have examined the expression pattern of SalAT in P.somniferum. SalAT was expressed in each major plant part analyzed—root,stem, leaf and capsule. This corresponds to the detection of transcriptof another morphine biosynthesis-specific gene, cor1, in each plantorgan analyzed (12). Additionally, salutaridinol 7-O-acetyltransferaseand codeinone reductase enzyme activity have each been detected in thecytosolic fraction of isolated latex (12,13). The gene cyp80b1participates in (S)-reticuline biosynthesis, occurring before abifurcation in the biosynthetic pathway that leads to more than 80isoquinoline alkaloids. Cyp80b1 is, therefore, common to severalbiosynthetic pathways including morphine, sanguinarine and noscapine.Transcript of cyp80b1 was also detected in all plant organs analyzed(12). Accumulation of morphinan alkaloids is thought to correlate withthe appearance of laticifer cells in the developing plant and indifferentiating plant cell culture (32,33). A reticulated laticifersystem associated with the vascular tissue is present through the aerialparts of the poppy plant. In roots, non-reticulated laticifers arepresent (34,35). The localization of three genes of morphinebiosynthesis, cyp80b1, salAT and cor1 is thus far consistent with theassumption this biosynthesis is, at least in part, associated withlaticifer cells. Interestingly, deacetylvindoline acetyltransferase hasbeen localized to laticifer cells in aerial parts of C. roseus (36).

[0049] The invention also relates to eukaryotic and prokaryotic cellstransformed or transfected by the nucleic acid sequences derived fromthe acetyltransferase gene. An example of a suitable prokaryotic cell isa bacterial cell. Examples of suitable eukaryotic cells are yeast cells,vertebrate cells such as mammalian cells, for example mouse, monkey, orhuman cells, or invertebrate cells such as insect cells. Plant cells areparticularly preferred. In the context of the present invention, theterm “plant” is to be understood as including mosses and liverworts. Theplant cells can be any type of plant cells, including monocotyledonousor dicotyledonous plant cells. The cells may be differentiated cells orcallus for example suspension cultures. Cells of the genus Papaver areparticularly preferred.

[0050] According to the invention, cells are transfected or transformedusing techniques conventional in the art, in conditions allowingexpression of the O-acetyltransferase or derivatives. A number oftransformation techniques have been reported for Papaver. For example,microprojectile bombardment of cell suspension cultures may be used(refs. 40, 41). Transformation may also be effected using Agrobacteriumtumefaciens (refs 42, 43), or Agrobacterium rhizogenes, (Refs 44, 45)using either cell suspension cultures or tissue explants. Internationalpatent application WO 9934663 also reports methods for transforming andregenerating poppy plants.

[0051] The cell type that is selected for transformation or transfectiondepends to a large extent upon the objective to be achieved. In fact,the nucleic acid molecules of the invention can be used to achieve anumber of objectives which will be discussed below. Depending on thetype of molecule introduced into the plant cell, and the metabolicpathways present in the cell, a wide range of effects can be achieved.

[0052] A first objective is to produce recombinant acetyltransferaseenzyme, or derivatives thereof. A preferred method for producingproteins having salutaridinol 7-O-acetyltransferase activity comprisesthe steps of:

[0053] i) transforming or transfecting a cell with a nucleic acidmolecule encoding enzymatically active salutaridinol7-O-acetyltransferase, in conditions permitting the expression of theprotein having salutaridinol 7-O-acetyltransferase activity,

[0054] ii) propagating the said cells, and

[0055] iii)recovering the thus-produced protein having salutaridinol7-O-acetyltransferase activity.

[0056] For the purpose of producing recombinant enzyme, any of the abovelisted cell-types can be used. Plant cells such as cells of a Papaverspecies, or insect cells, as demonstrated in the examples below, areparticularly suitable. Bacterial cells, such as E. coli, can also beused.

[0057] Nucleic acid constructs which encode enzymatically activesalutaridinol 7-O-acetyltransferase activity suitable for use in thismethod include the sequences illustrated in FIG. 9 (SEQ. ID No.13), FIG.10 (SEQ. ID No.15) and FIG. 11 (SEQ. ID No.17), degenerate equivalentsthereof, variants having at least 70% identity to the FIG. 10 (SEQ. IDNo.15) sequence, variants capable of hybridizing in stringent conditionsto the FIG. 10 (SEQ. ID No.15) sequence, and fragments thereof having alength of at least 1200, and preferably 1300 nucleotides. Therecombinant enzyme thus produced can be used in in vitro methods forproducing pentacyclic morphinan alkaloids, particularly thebaine.

[0058] A second, important aspect of the invention is a biotechnologicalproduction of thebaine, codeine and morphine. cDNAs encoding severalenzymes of morphine biosynthesis have now been isolated. The firstenzyme in the biosynthetic pathway for which a cDNA was isolated isnorcoclaurine 6-O-methyltransferase (37). The next is the cytochromeP-450-dependent monooxygenase (S)-N-methylcoclaurine 3′-hydroxylase(12,18). These enzymes are common to the morphine, noscapine andsanguinarine biosynthetic pathways. Specific to morphine biosynthesisare salutaridinol 7-O-acetyltransferase (reported herein) and codeinonereductase, the penultimate enzyme of the morphine pathway that reducescodeinone to codeine (13). A cDNA encoding an enzyme involved generallyin metabolism, but essential to the activity of the cytochromeP-450-dependent monooxygenase, the cytochrome P-450 reductase, has alsobeen isolated (38). Each of the cDNAs has been functionally expressed ininsect cell culture (S. frugiperda Sf9 cells) or in E. coli. Animmediate application of these cDNAs is in the metabolic engineering ofP. somniferum to obtain altered alkaloid profiles in the plant. Anothergoal is a biomimetic synthesis of morphinan alkaloids combiningchemically- and enzymatically-catalyzed steps. For this latterapplication, depending upon the plant-type used, additional cDNAsencoding enzymes that mediate transformations occurring between(R)-reticuline and morphine may need to be isolated and introduced.

[0059] This major objective of the invention thus relates to the use ofthe O-acetyltransferase genes and derivatives thereof to producepentacyclic morphinan alkaloids, particularly thebaine, in plants or inplant cell cultures, and to alter the alkaloid profiles ofalkaloid-producing plants such as poppies. In the context of theinvention, the term “alkaloid producing plant” signifies plants thatnaturally have the capacity to produce opium alkaloids, or morphinanalkaloids such as morphine, codeine, thebaine and oripavine.

[0060] For this objective, plant cells are used as host cells. For thisaspect of the invention, plants that are particularly preferred arethose belonging to the families Papaveraceae, Euphorbiaceae,Berberidaceae, Fumariaceae and Ranunculaceae, although other familiescan also be used. These families are particularly advantageous becausethey share at least partially, P. somniferum's biosynthetic pathwayleading from (R)-reticuline to morphine (3). This pathway is representeddiagramatically below:

[0061] For the production of pentacyclic morphinans, particularythebaine, nucleic acid molecules encoding proteins having salutaridinol7-O-acetyltransferase activity are introduced into plant cells whichnaturally already have the capacity to produce (R)-reticuline, andpreferably also to produce salutaridine and/or salutaridinol. Suchplants are preferred because they are highly likely to have theendogenous enzymes necessary to carry out the complete pathway from(R)-reticuline to thebaine.

[0062] Tables 2A, 2B, 2C and 2D below provide non-limiting examples ofplants able to produce these different products from (R)-Reticuline. Inthese Tables, ‘+’ provides a non-quantitative indication of the capacityto produce the indicated compound, ‘−’ indicates an inability to producethe indicated compound at detectable levels, and * indicates tracelevels, depending on the sensitivity of the analysis (Ref. 39): TABLE 2APlants of the genus Papaver producing (R) - reticuline, salutaridine,thebaine and other pentacyclic alkaloids (R) - Plant Ret. SalutaridineThebaine Codeine Morphine P. bracteatum + + + + * P.cylindricum + + + + + P. orientale + + + + * P. setigerum + + + + + P.somniferum + + + + +

[0063] TABLE 2B Plants of the genus Papaver producing (R) - reticuline,salutaridine, thebaine Plant (R) - Ret. Salutaridine Thebaine CodeineMorphine P. pseudo- + + + − − orientale P. lauricola + + + − − P.persicum + + + − − P. caucasium + + + − − P. carmeli + + + − −

[0064] TABLE 2C Plants producing (R) - reticuline and salutaridine Plant(R) - Ret. Salutaridine Thebaine Codeine Morphine P. acrochaetum + + − −− P. alpinum + + − − − P. armeniacum + + − − − P. atlanticum + + − − −P. aurantiacum + + − − − P. corona + + − − − P. croceum + + − − − P.curviscapum + + − − − P. degenii + + − − − P. ernesti + + − − − P.fugax + + − − − P. gracile + + − − − P. heldreichii + + − − − P.kerneri + + − − − P. lasiothrix + + − − − P. nudicaule + + − − − P.pilosum + + − − − P. polychaetum + + − − − P. rhaeticum + + − − − P.rubroaurantiacum + + − − − P. sendtneri + + − − − P. strictum + + − − −P. tartricum + + − − − P. tauricola + + − − − C. campestris ¹ + + − − −C. balsamifera + + − − − C. ferruginellus + + − − − C. ruizianus + + − −−

[0065] TABLE 2D Plant families producing (R)-Reticuline Plant family(R)-Reticuline Berberidaceae + e.g. Berberis spp. Podophyllum spp.Fumariaceaeae + e.g. Adlumia spp. Cordyalis spp. Dicentra spp. Fumariaspp. Papaveraceae + e.g. Papaver spp. Argemone spp. Bocconia spp.Glaucium spp. Eschscholtzia spp. Ranunculaceae + e.g. Thalictrum spp.

[0066] According to a preferred variant, for the production ofpentacyclic morphinans such as thebaine, morphine and codeine, a hostplant cell is selected that naturally contains a gene encodingsalutaridinol 7-O-acetyltransferase. Such plants may be identified inseveral ways:

[0067] genomic DNA, cDNA or mRNA of the plant hybridises in stringentconditions to the nucleic acid molecule illustrated in FIG. 10 (SEQ. IDNo.15), or a fragment or variant thereof, and/or

[0068] the plant is capable of producing thebaine, and possibly otherpentacyclic morphinan alkaloids such as morphine and codeine, and/or

[0069] salutaridinol 7-O-acetyltransferase activity can be detected inthe latex of the plant.

[0070] Specific examples of such plants are shown in Tables 2A and 2Babove. The endogenous salutaridinol-7-O-acetyltransferase activity issupplemented by the introduction of an exogenous nucleic acid moleculeencoding a protein having salutaridinol-7-O-acetyltransferase activity.The expression of the exogenous acetyltransferase leads toover-expression of the enzyme, and increases thebaine production. Thenatural alkaloid profile of the plant is thereby altered. Suchalteration can take the form of an alteration in total alkaloid yield,or in the type of alkaloid, or in the relative proportions of differentalkaloids, produced by the plant. For example, members of the genusPapaver, e.g. P. somniferum, can be altered using the process of theinvention to produce thebaine as major or sole alkaloid.

[0071] In general, for the production of morphine and codeine, plantsare preferred which have all the necessary endogenous enzymes i.e.plants that naturally produce morphine and codeine, for example thoseshown in Table 2A.

[0072] For the production of thebaine, it is similarly possible to use aplant cell that naturally produces substantial amounts of thebaine,whereby the thebaine produced is the result of an over-expression ofacetyltransferase from both the endogenous and exogenous genes. Examplesare given in Table 2A and 2B. It is however possible, for thebaineproduction, to use a cell of a plant that does not naturally producesubstantial amounts of thebaine. According to this latter embodiment,the exogenous salutaridinol 7-O-acetyltransferase confers upon the plantor plant cell the ability to synthesize thebaine. Examples of plantswhich can be used in this variant of the invention are shown in Tables2C and 2D.

[0073] As particularly preferred plants for this embodiment of theinvention, members of the Papaveraceae family, particularly Papaversomniferum, Papaver bracteatum, Papaver setigerum, Papaver orientate,Papaver pseud-orientale, Papaver cylindricum can be cited.

[0074] According to a further aspect of the invention, the alkaloidprofile of an alkaloid-producing plant (such as the Papaveraceae) can bealtered by introducing nucleic acid molecules which have inhibitoryactivity on salutaridinol-7-O-acetyltransferase expression, for examplemolecules which are complementary to the deacetylase gene or itstranscript. Antisense molecules complementary to the transcript of thesequence illustrated in FIG. 10 (SEQ. ID No.15), or a ribozyme capableof hybridising to the said transcript are examples of such inhibitorymolecules. Such molecules have the capacity to inhibit functionalexpression of the endogenous salutaridinol-7-O-acetyltransferase, thusreducing thebaine production. The altered thebaine production results ina global modification of the alkaloid profile of the plant.

[0075] As part of the process of production of morphinans, (e.g.morphine, codeine or thebaine), the transformed or transfected cells arepropagated to produce a multiplicity of morphinan-producing cells, andthen conventional techniques are used to recover the pentacyclicalkaloid(s). The multiplicity of cells produced by propagation may be acell culture of differentiated or undifferentiated cells, for examplecallus suspension cultures. Alternatively the cells may be regeneratedto provide a whole transgenic or chimeric plant. The invention alsoencompasses the cell cultures and transgenic plants produced from thetransformed or transfected cells. Particularly preferred are transgenicplants of the genus Papaver, for example those in Tables 2A and 2B,which exhibit over-expression of salutaridinol 7-O-acetyltransferase.These plants are characterised by the presence of at least oneendogenous salutaridinol 7-O-acetyltransferase gene, accompanied by atleast one copy of an exogenous salutaridinol 7-O-acetyltransferase geneof the invention. Typically the exogenous SalAT gene can bedistinguished from the endogenous gene by the presence of heterologoustranscription regulatory sequences.

[0076] Other preferred transgenic plants exhibit reduced expression ofsalutaridinol 7-O-acetyltransferase as a result of the introduction of anucleic acid encoding a salutaridinol 7-O-acetyltransferase inhibitor,for example a ribozyme or an antisense molecule.

[0077] The invention also relates to the seed of the transgenic plantsof the invention, and also to the opium and straw, or straw concentratesproduced by the altered plants.

[0078] The morphine biosynthetic genes of the invention permitinvestigation of the question of why only P. somniferum producesmorphine, while other Papaver species such as P. rhoeas, P. orientate,P. bracteatum, P. nudicaule and P. atlanticum do not. SalAT transcriptwas detected in RNA isolated from P. somniferum, P. orientate and P.bracteatum, but not in RNA from P. nudicaule and P. atlanticum. This isconsistent with the expected distribution based upon accumulation ofalkaloids having the morphinan nucleus in these species (i.e. morphinein P. somniferum, thebaine in P. bracteatum and oripavine in P.orientate). This is in sharp contrast to those results obtained for cor1transcript, which was detected also in Papaver species that are notknown to accumulate codeine (12). The genes of alkaloid biosynthesis inP. somniferum will certainly continue to provide useful information onthe molecular evolution of plant secondary metabolism in latex systems.

[0079] Various aspects of the invention are illustrated in the Figures:

[0080]FIG. 1. Schematic biosynthetic pathway leading from salutaridinolto morphine in opium poppy. The 7-hydroxy moiety of salutaridinol isactivated by the transfer of an acetyl group from acetyl CoA, catalyzedby salutaridinol 7-O-acetyltransferase. Elimination of acetate to formthebaine is a pH-dependent reaction that can proceed spontaneously. Thedemethylation of thebaine and codeine are each thought to be catalyzedby cytochrome P-450-dependent enzymes.

[0081]FIG. 2. Amino acid sequence comparison of salutaridinol7-O-acetyltransferase to other plant acetyltransferases involved insecondary metabolism. SALAT, salutaridinol 7-O-acetyltransferase from P.somniferum (this work); DAT, deacetylvindoline acetyltransferase of C.roseus (22); BEAT, benzylalcohol acetyltransferase from Clarkia breweri(26), HCBT, anthranilate N-hydroxycinnamoyl/benzoyltransferase fromDianthus caryophyllus (27); DBAT, 10-deacetylbaccatinIII-10-O-acetyltransferase and TAT, taxadienol acetyltransferase, bothfrom Taxus cuspidata (28,29). Black boxes indicate conserved residues;white boxes indicate the internal peptide sequences obtained from nativesalutaridinol 7-O-acetyltransferase; arrows indicate the positions ofthe peptides used to design oligodeoxynucleotide primers for RT-PCR; #denotes positions of the highly conserved consensus sequence HXXXD (SEQ.ID Nos.7 to 12).

[0082]FIG. 3. Genomic DNA gel blot analysis of the salutaridinol7-O-acetyltransferase gene in opium poppy. Genomic DNA isolated from P.somniferum 3-week-old seedlings was hybridized to salAT full-length cDNAand was visualized by phosphorimagery. The number of restrictionendonuclease recognition sites that occur within the open reading frameare as follows: EcoRI, 0; HindIII, 0; ApoI, 1; SalI, 1; SpeI, 1; HincII,I; MspI, 3.

[0083]FIG. 4. RNA gel blot analysis of A) salAT is expressed in lane 1.root, lane 2. capsule, lane 3 stem and lane 4 leaf of the mature poppyplant and B) salAT transcript accumulation in 3-week-old seedlings oflane 1. P. somniferum, lane 2. P. orientale, lane 3. P. atlanticum, lane4. P. nudicaule, and lane 5. P. bracteatum. The dark portion of eachpanel is a photograph of ethidium bromide visualized RNA in the gelprior to blotting. This serves as an RNA loading control. The bottomportion of each panel is the results obtained after blotting andhybridization to salAT full-length cDNA visualized by phosphorimagery.

[0084]FIG. 5. SDS-PAGE analysis of fractions from the purification ofrecombinant salutaridinol 7-O-acetyltransferase from S. frugiperda Sf9cell culture medium. Lane, 1. protein standards (MBI Fermentas), lane 2.250 mM imadazole buffer elution of salutaridinol 7-O-acetyltransferasefrom the Talon resin, lane 3. 10 mM imadazole buffer wash of Talonresin, lane 4. Talon column flow-through, lane 5. Sf9 cell culturemedium after ammonium sulfate precipitation and dialysis.

[0085]FIG. 6. TLC radio-chromatogram of an aliquot of an enzyme assaycontaining A) [7-³H]salutaridinol, acetyl CoA and boiled enzyme, B)[7-³H]salutaridinol, acetyl CoA and recombinant salutaridinol7-O-acetyltransferase and C) [7-³H]thebaine standard. D) HPLC-positiveion electrospray mass spectral analysis of the product produced in anassay containing salutaridinol, acetyl CoA and recombinant salutaridinol7-O-acetyltransferase. E) HPLC-positive ion electrospray mass spectralanalysis of thebaine standard.

[0086]FIG. 7: Table 1 as referred to in the Examples.

[0087]FIG. 8: Deduced amino acid sequence of P. somniferum salutaridinol7-O-acetyltransferase. An identical match was observed between thededuced and directly determined amino acid sequences of ten internalpeptides distributed throughout the open reading frame (SEQ. ID No.14).

[0088]FIG. 9: cDNA sequence of P. somniferum salutaridinol7-O-acetyltransferase (1785 nucleotides). The start codon is at position166, and the Stop at position 1588 (SEQ. ID No.13).

[0089]FIG. 10: Coding sequence of P. somniferum salutaridinol7-O-acetyltransferase, showing the amino acid sequence also (SEQ. IDNo.15).

[0090]FIG. 11: Alignment of cDNA sequences of P. somniferumsalutaridinol 7-O-acetyltransferase from different sources. The top lineshows cDNA cloned from P. somniferum cultivar CO48 (designated herein asSalAT 2 or “SAT 2 CO48” in FIG. 11 (SEQ. ID No.17)). The bottom lineshows cDNA cloned from P. somniferum cell suspension cultures(designated herein as SalAT 1 or “SAT 1 Halle” in FIG. 11). Differencesin nucleotide sequence are shown in bold type and changes in amino acidsequence are above and below the nucleic acid sequences (SEQ. ID No.17)

[0091] FIG.12: The pPLEX X002 binary plasmid used in the transformationof SalAT 2 into poppy explants. The SalAT 2 cDNA was introduced intopPLEX X002 at the multiple cloning site between the S4S4 promoter andthe Me1 terminator.

EXAMPLES

[0092] In the following Examples, salutaridinol 7-O-acetyltransferase[EC 2.3.1.150] has been characterized by purifying the native enzyme toapparent homogeneity, and determining amino acid sequences for internalpeptides. A cDNA clone was then generated by RT-PCR using P. somniferummRNA as template. Heterologous expression in a baculovirus vector ininsect cells yielded functional enzyme that acetylated the 7-hydroxylmoiety of salutaridinol in the presence of acetyl CoA. Enzymicproperties were determined for the recombinant protein. The apparentK_(m) value for salutaridinol was determined to be 9 μM, and 54 μM foracetyl CoA.

[0093] An identical match was observed between the deduced (FIG. 8 (SEQ.ID No.14)) and directly determined amino acid sequences of ten internalpeptides distributed throughout the open reading frame. The calculatedmolecular mass of the enzyme is 52.6 kDa, which is consistent with theapparent molecular mass of 50 kDa determined by SDS-PAGE (8). The aminoacid sequence of salutaridinol 7-O-acetyltransferase is most similar(37% identity) to that of deacetylvindoline acetyltransferase ofCatharanthus roseus

[0094] The results obtained by RACE-PCR indicated that the reading frameis 1425 nucleotides long corresponding to 474 amino acids (FIG. 10 (SEQ.ID No.15)). These values correlate well with the transcript sizeobtained by RNA gel blot analysis. The full length cDNA is illustratedin FIG. 9 (SEQ. ID No.13). Gene transcript was detected in extracts fromP. orientale and P. bracteatum, in addition to P. somniferum. GenomicDNA gel blot analysis indicated that there is likely a single copy ofthis gene in the P. somniferum genome.

[0095] The abbreviations used are: RT-PCR, reverse transcriptasepolymerase chain reaction; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; HPLC, high performance liquidchromatography; RACE, rapid amplification of cDNA ends; HPLC-MS, highperformance liquid chromatography mass spectrometry; SalAT, cDNAencoding salutaridinol 7-O-acetyltransferase; TLC, thin layerchromatography; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; cor1,cDNA encoding codeinone reductase; cyp80b1, (S)-N-methylcoclaurine3′-hydroxylase; DEPC, diethylpyrocarbonate

[0096] I. Experimental Procedures

[0097] Plant Material: Cultured suspension cells of opium poppy Papaversomniferum were provided by the cell culture laboratories of theLehrstuhl für Pharmazeutische Biologie and of the Leibniz-Institut fürPflanzenbiochemie. Cultures were routinely grown in 1 liter conicalflasks containing 400 ml of Linsmaier-Skoog medium (14) over 7 days at23° C. on a gyratory shaker (100 rpm) in diffuse light (750 lux).Differentiated P. somniferum, P. bracteatum, P. orientale, P. nudicaule,P. atlanticum, P. rhoeas and Chelidonium majus plants were grownoutdoors in Upper Bavaria or in Saxony-Anhalt. P. somniferum ssp.setigerum plants were grown in a greenhouse at 24° C., 18 h light and50% humidity.

[0098] Purification of Native Enzyme and Amino Acid Sequence Analysis:Salutaridinol acetyltransferase was purified from P. somniferum cellsuspension cultures exactly according to Lenz and Zenk (8). The purifiedenzyme preparation was subjected to SDS-PAGE to remove traces ofimpurities and the Coomassie brilliant blue R-250-visualized bandrepresenting the acetyltransferase was digested in situ withendoproteinase Lys-C as previously reported (15,16). The peptide mixturewas resolved by reversed phase HPLC (column, Merck Lichrospher RP18; 5μm (4×125 mm); solvent system (A) 0.1% trifluoroacetic acid (B) 0.1%trifluoroacetic acid/60% acetonitrile; gradient of 1% per min; flow rateof 1 ml min⁻¹) with detection at 206 nm. Microsequencing of ten of thepeptides was accomplished on an Applied Biosystems model 470 gas-phasesequencer.

[0099] Generation of Partial cDNAs from P. somniferum: Partial cDNAsencoding salutaridinol acetyltransferase from P. somniferum wereproduced by PCR using cDNA generated by reverse transcription of mRNAisolated from 7-day-old suspension cultured cells. DNA amplificationusing either Taq or Pfu polymerase was performed under the followingconditions: 3 min at 94° C., 35 cycles of 94° C., 30 s; 50° C., 30 s;72° C., 1 min. At the end of 35 cycles, the reaction mixtures wereincubated for an additional 7 min at 72° C. prior to cooling to 4° C.The amplified DNA was resolved by agarose gel electrophoresis, the bandsof approximately correct size (537 bp) were isolated and subcloned intopGEM-T Easy (Promega) prior to nucleotide sequence determination. Thespecific sequences of the oligodeoxynucleotide primers used are given inthe Results section.

[0100] Generation of Full-Length cDNAs: The sequence informationrequisite to the generation of a full-length cDNA was derived from thenucleotide sequence of the partial cDNA produced as described in theResults section. The complete nucleotide sequence was generated in twosteps using one salutaridinol acetyltransferase-specific PCR primer(5′-GCC GCA GGC CAA CAA GGG TTG AGG TGG-3′ (SEQ. ID No.2) for 5′-RACEand 5′-CCC ATC CTG CAC CAG CTA CTT ATC C-3′ (SEQ. ID No.1) for 3′-RACE)and one RACE-specific primer as specified by the manufacturer. The 5′-and 3′-RACE-PCR experiments were carried out using a Marathon cDNAamplification kit (Clontech). RACE-PCR was performed using the followingPCR cycle: 3 min at 94° C., 35 cycles of 94° C., 30 s; 60° C., 30 s; 72°C., 2 min. At the end of 35 cycles, the reaction mixtures were incubatedfor an additional 7 min at 72° C. prior to cooling to 4° C. Theamplified DNA was resolved by agarose gel electrophoresis, the bands ofthe expected size (1265 bp for 5′-RACE and 917 bp for 3′-RACE) wereisolated and subcloned into pGEM-T Easy prior to sequencing.

[0101] The full-length clone was generated in one piece using theprimers 5′-CCA TGG CAA CAA TGT ATA GTG CTG CT-3′ (SEQ. ID No.3) and5′-AGA TCG AAT TCA ATA TCA AAT CAA TTC AAG G-3′ (SEQ. ID No.4) for PCRwith P. somniferum cell suspension culture cDNA as template. The finalprimers used for cDNA amplification contained recognition sites for therestriction endonucleases NcoI and EcoRI, appropriate for subcloninginto pFastBac HTa (Life Technologies) for functional expression. DNAamplification was performed under the following conditions: 3 min at 94°C., 35 cycles of 94° C., 30 s; 60° C., 30 s; 72° C., 2 min. At the endof 35 cycles, the reaction mixtures were incubated for an additional 7min at 72° C. prior to cooling to 4° C. The amplified DNA was resolvedby agarose gel electrophoresis, the band of approximately correct size(1440 bp) was isolated and subcloned into pCR4-TOPO (Invitrogen) priorto nucleotide sequence determination.

[0102] Heterologous Expression and Enzyme Purification: The full-lengthcDNA generated by RT-PCR was ligated into pFastBac HTa that had beendigested with restriction endonucleases NcoI and EcoRI. The recombinantplasmid was transposed into baculovirus DNA in the Escherichia colistrain DH10BAC (Life Technologies) and then transfected into Spodopterafrugiperda Sf9 cells according to the manufacturer'instructions. Theinsect cells were propagated and the recombinant virus was amplifiedaccording to (17,18). INSECT-XPRESS serum-free medium (Bio Whittaker)was used in the enzyme expression experiments.

[0103] After infection of 150 ml suspension grown insect cells hadproceeded for 3-4 days at 28° C. and 130 rpm, the cells were removed bycentrifugation under sterile conditions at 1000×g for 10 min at 4° C.All subsequent steps were performed at 4° C. The pellet was discardedand the medium was slowly brought to 80% saturation with ammoniumsulfate under constant slow stirring. The precipitated proteins werecollected by centrifugation at 10,000×g for 30 min at 4° C. The pelletwas dissolved in a minimal volume of 0.5 M NaCl, 10 mMbeta-mercaptoethanol, 2.5 mM imidazole, 20 mM Tris-HCl adjusted finallyto pH 7.0 and was dialyzed for 12-16h against this same buffer. TheHis-tagged salutaridinol acetyltransferase was purified by affinitychromatography using a cobalt resin (Talon, Clontech) according to themanufacturer'instructions.

[0104] Enzyme Assay and Product Identification: The acetylationcatalyzed by salutaridinol acetyltransferase was assayed according toLenz and Zenk (8). The reaction mixture was extracted once with 1 volumeCHCl₃ and was resolved by TLC (plates, silica gel 60 F₂₅₄, Merck;solvent system, chloroform:acetone:diethylamine (5:4:1)). Theradioactivity present on the TLC plates was localized and quantitatedwith a Rita Star TLC scanner (Raytest). The identity of the enzymicreaction product as thebaine was ascertained by HPLC-MS using a FinniganMAT TSQ 7000 (electrospray voltage 4.5 kV; capillary temperature 220°C.; carrier gas N₂) coupled to a Micro-Tech Ultra-Plus Micro-LC equippedwith an Ultrasep RP18 column; 5 μm; 1×10 mm). Solvent system (A) 99.8%(v/v) H₂O, 0.2% HOAc (B) 99.8% CH₃CN (v/v), 0.2% HOAc; gradient: 0-15min 10-90% B, 15-25 min 90% B; flow 70μl min⁻¹). The positive ionelectrospray (ES) mass spectrum for thebaine (retention time 17.4±0.1min; m/z=312) was characteristic of the standard reference compound.

[0105] General Methods: Latex was collected and resolved as previouslydescribed (19,20). Low molecular weight compounds were removed from thesupernatant of the resolved latex by passage through a PD 10 column into20 mM Tris, 10 mM-mercaptoethanol, pH 7.5 (Amersham Pharmacia). TotalRNA was isolated and RNA gels were run and blotted as describedpreviously (18). Genomic DNA was isolated and DNA gels were run andblotted according to (21). cDNA clones were labeled by PCR labeling with[alpha-³²P]dATP. Hybridized RNA on RNA gel blots and DNA on DNA gelblots were visualized with a STORM phosphor imager (Molecular Dynamics).The entire nucleotide sequence on both DNA strands of the full-lengthclone was determined by dideoxy cycle sequencing using internal DNAsequences for the design of deoxyoligonucleotides as sequencing primers.Saturation curves and double reciprocal plots were constructed with theFig. P program Version 2.7 (Biosoft, Cambridge, UK). The influence of pHon enzyme activity was monitored in sodium citrate (pH 4-6), sodiumphosphate (pH 6-7.5), Tris-HCl (pH 7.5-9), glycine/NaOH (pH 9-10.5) andCAPS (pH 10-12) buffered solutions.

[0106] II. Results

[0107] Purification and Amino Acid Sequence Analysis of Salutaridinol7-O-Acetyltransferase-Salutaridinol 7-O-acetyltransferase was purifiedto apparent electrophoretic homogeneity from opium poppy cell suspensioncultures and the amino acid sequence of ten endoproteinaseLys-C-generated peptides was determined. The sequences and relativepositions of these internal peptides are indicated by unshaded boxes inFIG. 2 (SEQ. IDs Nos. 7 to 12). A comparison of these amino acidsequences with those available in the GenBank/EMBL sequence databasesindicated no relevant similarity to known proteins. PCR primer pairsbased on a series of salutaridinol 7-O-acetyltransferase peptidecombinations also yielded only DNA fragments of irrelevant sequence.

[0108] Isolation of the cDNA Encoding Salutaridinol7-O-Acetyltransferase: During the course of the initial RT-PCRexperiments, sequence comparison information appeared in the literaturefor another acetyltransferase of plant alkaloid biosynthesis (22). Thetranslation of the sequence of the cDNA encoding deacetylvindoline4-O-acetyltransferase was homologous to a series of other putative plantacetyltransferases. A conserved region near the carboxy terminus of theproteins was used to design a degenerate antisense oligodeoxynucleotideprimer for PCR. The sense primer was based upon an internal peptidesequence of salutaridinol 7-O-acetyltransferase. The primer sequenceswere as follows: Sense Primer (FVFDFAK): 5′ TTT/C GTG/A/T TTT/C GAC/TTTT/C GCA/T AA 3′ (SEQ. ID No.5) Antisense Primer (DFGWG motif):5′ A/C/G/TGG C/TTT A/C/G/TCC CCA A/C/G/TCC C/AAA A/GTC 3′ (SEQ. ID No.6)

[0109] The positions of these peptides are indicated by arrows in FIG. 2(SEQ. IDs Nos. 7 to 12). RT-PCR performed with this primer pair yieldeda DNA product of the correct size and sequence for the opium poppyacetyltransferase. RACE-PCR was then used to generate each the 5′- and3′-portions of the cDNA using nondegenerate nucleotide sequenceinformation provided from the original PCR product.

[0110] Sequence Analysis of pSalAT: Translation of the completenucleotide sequence of salAT yielded a polypeptide of 474 amino acidscontaining no apparent signal peptide. This is consistent with thecytosolic localization of the enzyme activity (6). The enzyme activityis also operationally found associated with the cytosolic fraction ofexuded latex. The salAT amino acid sequence contains residues conservedin other plant acetyltransferases as indicated by the black boxes inFIG. 2 (SEQ. IDs No.7 to 12). The longest contiguous region of conservedamino acids are the five residues DFGWG near the carboxy terminus thatwere used for primer design and are indicated by an arrow. Conservedhistidine and aspartate residues (HXXXD; denoted by # in FIG. 2) thoughtto be involved in catalysis as characterized by x-ray crystallographyfor the bacterial enzymes chloramphenicol acetyltransferase anddihydrolipoamide acetyltransferase are also present in salutaridinol7-O-acetyltransferase (23,24). Covalent modification of salutaridinol7-O-acetyltransferase by treatment with diethylpyrocarbonate (DEPC)resulted in the inhibition of enzyme activity (50% inhibition at 3 mMDEPC; 92% inhibition at 5 mM DEPC) (25). The inactivation by 5 mM DEPCcould be reduced from 92% to 46% by preincubation of the enzyme with 30mM acetyl CoA.

[0111] The amino acid sequence of salutaridinol 7-O-acetyltransferase ismost similar (37% identity) to that of deacetylvindolineacetyltransferase of C. roseus (22). Other similar plantacyltransferases involved in secondary metabolism are benzylalcoholacetyltransferase from Clarkia breweri (34%) (26), anthranilateN-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus (25%)(27), taxadienol acetyltransferase (24%) and 10-deacetylbaccatinIII-10-O-acetyltransferase (22%), both from Taxus cuspidata (28,29).

[0112] Genomic DNA and Gene Expression Analysis: A genomic DNA gel blotanalysis of salAT in P. somniferum is presented in FIG. 3. Therestriction endonucleases ApoI, SalI, SpeI and HincII each recognize onehydrolysis site within the salAT open reading frame, yielding twohybridizing bands on the Southern blot. There are no recognition sitesfor HindIII in the open reading frame. Correspondingly, only a singleband hybridizes, but it is of approximately one half the predictedlength. This indicates the possible presence of a small intron in thegene. Three recognition sites are present for MspI, theoreticallyresulting in four hybridizing DNA fragments. Two hybridizing bands ofpredictable length should have been present, one at 180 and 511 bp. Theabsence of these two bands also indicates that intron(s) may be presentin the gene. No recognition site is present for EcoRI in the openreading frame, but two hybridizing bands are present on the gel blot,also suggesting an intron, which contains an EcoRI restriction site. Amore thorough analysis of this point awaits isolation of a genomicclone. These results, taken together, support a single gene hypothesis,but do not exclude two very similar, clustered alleles.

[0113] This is in stark contrast to the other known morphine-specificbiosynthetic gene cor1 encoding codeinone reductase, for which at leastsix alleles are expressed (13). RNA gel blot analysis suggests that, asfor cor1, salAT is expressed in root, stem, leaf and capsule of themature poppy plant (FIG. 4A) (12,13). There appears to be noorgan-specific expression of either of these morphine biosyntheticgenes. Analysis of RNA from several members of the genus Papaverdemonstrates salAT transcript accumulation in three-week-old seedlingsof P. orientate and P. bracteatum, though not in P. atlanticum or P.nudicaule (FIG. 4B). P. orientate accumulates the alternate biosyntheticprecursor oripavine and P. bracteatum accumulates the morphinebiosynthetic precursor thebaine, both of which structures contain theoxide bridge formed by action of salutaridinol 7-O-acetyltransferase. Itwas, therefore, expected that these two species should containhybridizing salAT transcript. Neither P. atlanticum nor P. nudicaulecontain an alkaloid with the morphinan skeleton, consistent with theabsence of transcript in these two species.

[0114] Purification and Functional Characterization of RecombinantEnzyme: The salAT cDNA was constructed to express the recombinantprotein with six histidine residues elongating the amino terminus. Theprotein was then purified from Spodoptera frugiperda Sf9 cell culturemedium in two steps (ammonium sulfate precipitation/dialysis, cobaltaffinity-chromatography) to yield electrophoretically homogeneous enzymewith an overall yield of 25% and 22-fold purification (FIG. 5). Perliter, the insect cell culture typically produced 2.0 mg (150 nmol s⁻¹)of recombinant enzyme.

[0115] Radioassay of pure, recombinant enzyme using [7-³H]salutaridinolas substrate resulted in 100% conversion into a product that co-migratedduring TLC with authentic thebaine standard (FIG. 6A-C). The positiveion electrospray mass spectrum of the enzymic product produced whensalutaridinol was used as substrate correlated well with that ofthebaine standard (FIG. 6D,E). The apparent K_(m) value forsalutaridinol was determined to be 9 μM at a fixed concentration ofacetyl CoA of 30 mm. The apparent K_(m) value for acetyl CoA wasdetermined to be 54 μM at a fixed concentration of salutaridinol of 10mM. The V_(max) for the acetylation of salutaridinol was 25 pmol s⁻¹with a temperature optimum of 47° C. and a pH optimal range of 7-9 understandard assay conditions. The recombinant enzyme acetylated7(S)-salutaridinol and nudaurine (apparent K_(m) nudaurine 23 μM at 30mM acetyl CoA, apparent K_(m) acetyl CoA 106 μM at 10 mM nudaurine,V_(max) 19 pmol s⁻¹) at C-7, but not 7(R)-salutaridinol, salutaridine,codeine, morphine or deacetylvindoline. The kinetic values and chemicalstructures for the alkaloidal substrates salutaridinol and nudaurine aresummarized in Table I. As designated by the ratio k_(cat)/K_(m)(salutaridinol):k_(cat)/K_(m)(nudaurine), the enzyme acetylatessalutaridinol preferentially to nudaurine by a factor of 3.3.

[0116] Strain Deposit:

[0117] The cDNA encoding Salutaridinol 7-O-Acetyltransferase from P.somniferum (reading frame only, 1425 nucleotides as shown in FIG. 10(SEQ. ID No.15)) has been deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ) on 6 ^(th) June 2002 underAccession number DSM 15044. The SalAT cDNA, carried in vectorpCRT7/NT-TOPO in E.coli strain BL21DE3, is under the control of a T7promoter. The plasmid further carries an ampicillin resistance gene.

[0118] Cloning of a Variant of SalAT 1 and its Transformation intoPoppy.

[0119] The cDNA encoding SalAT 1 described above has enabled furtherSalAT sequences to be cloned from different P. somniferum lines. Theseresults provide evidence of natural genetic variation.

[0120] A new cDNA was cloned from messenger RNA isolated from poppycultivar CO48 (Papaver somniferum). Reverse transcriptase PCR usingprimers designed to the 5′ and 3′ UTR regions of the SalAT 1 sequence(FIG. 9 (SEQ. ID No.13)), amplified a product of the predicted size fromCO48 which was cloned into pGEM-Teasy. The primers had the followingsequences: Forward primer: CCTCGAGCCA TTATCAATCC TGTTAAACAG TTAAACACSAT_126F_XhoI (SEQ. ID No.19) Reverse primer: CCCTAGGGGA AATGGAGAAAATCATGATTA CGCAACAC SAT_1639R_AvrII (SEQ. ID No.18)

[0121] The primer SAT_(—)126F_XhoI places a XhoI site on the end of thePCR product, and SAT_(—)1639R_AvrII places an AvrII site on the end ofthe PCR product. This facilitates cloning first into pGEMT and thenpPLEX.

[0122] Two independent RT PCR clones were sequenced. These two wereidentical to each other in sequence and are referred to as SAT2 CO48 (orSalAT 2). The SAT2 CO48 (SalAT 2) had an intact ATG for the start oftranslation and it differed from the original SalAT 1 clone in 6nucleotides. The new CO48 cDNA (SalAT 2) sequence is illustrated in FIG.11 (SEQ. ID No.17) and compared to the original clone. The two clonesare in-frame throughout but there were 6 nucleotide differencesresulting in 5 amino acid changes as shown.

[0123] The SAT2 CO48 (SalAT 2) cDNA was cloned into pPLEX X002 (FIG. 12)between the S4S4 promoter and the Me1 terminator. The S4S4 doublepromoter derives from subterranean clover stunt virus segment 4 (Boevinket al, 1995). The Me1 terminator derives from Flaveria bidentis malicenzyme gene (Marshall et al, 1997).

[0124] Clones were sequenced to verify sequence integrity. Thetransformation binary pPLEX X002-SAT was transformed into Agrobacteriumtumefaciens strain Agl1 (Lazo et al, 1991). Sequencing verified theSalAT remained intact and unchanged after the transformation intoAgrobacterium.

[0125] This was used to transform hypocotyl pieces of TasmanianAlkaloids poppy cultivar CO58-34 (P. somniferum). The method used was asdescribed in patent application “Methods for plant transformation andregeneration” [WO9934663]. Seedling hypocotyl pieces were incubated in asuspension of the Agrobacterium for 10-15 minutes. Explants were thentransferred to medium B50medium consisting of B5 macronutrients,micronutrients, iron salts and vitamins (Gamborg et al, 1968), 20 g.L⁻¹sucrose using 0.8% Agar, 1 mg.L⁻¹ 2,4-dichlorophenoxy acetic acid(2,4-D) and 10 mM MES buffer. The pH was adjusted with 1M KOH to pH 5.6.

[0126] After four to five days co-cultivation explants were washed insterile distilled water, until the water was clear of evidentAgrobacterial suspension, blotted on sterile filter paper andtransferred to the same medium but contained 150 mg.L⁻¹ Timentin (toselect against the Agrobacterium) and 25 mg.L⁻¹ paromomycin (to selectfor transformed plant cells). Explants were transferred to fresh mediumof the same composition including antibiotic selection agents, everythree weeks.

[0127] Explants initially produced transgenic translucent brownishcallus consisting of large cells. This was termed type I callus. Thetransformed nature of the callus was demonstrated by growth on selectivemedium. Subsequently they formed small regions of white, compactembryogenic transgenic callus usually at about 7-8 weeks, and this wastermed type II callus. Transgenic somatic embryos develop on this callusafter 3-6 weeks and plantlets develop from these embryos and aretransferred to soil.

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1 19 1 24 DNA Artificial Sequence Description of Artificial Sequence XhoI primer 1 cccatcctgc accagctact tatc 24 2 27 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 2 gccgcaggcc aacaagggttgaggtgg 27 3 26 DNA Artificial Sequence Description of ArtificialSequence PCR primer 3 ccatggcaac aatgtatagt gctgct 26 4 31 DNAArtificial Sequence Description of Artificial Sequence PCR primer 4agatcgaatt caatatcaaa tcaattcaag g 31 5 20 DNA Artificial SequenceDescription of Artificial Sequence sense primer 5 ttygtdttyg ayttygcwaa20 6 21 DNA Artificial Sequence Description of Artificial Sequenceantisense primer 6 nggyttnccc canccraart c 21 7 474 PRT Papaversomniferum 7 Met Ala Thr Met Tyr Ser Ala Ala Val Glu Val Ile Ser Lys GluThr 1 5 10 15 Ile Lys Pro Thr Thr Pro Thr Pro Ser Gln Leu Lys Asn PheAsn Leu 20 25 30 Ser Leu Leu Asp Gln Cys Phe Pro Leu Tyr Tyr Tyr Val ProIle Ile 35 40 45 Leu Phe Tyr Pro Ala Thr Ala Ala Asn Ser Thr Gly Ser SerAsn His 50 55 60 His Asp Asp Leu Asp Leu Leu Lys Ser Ser Leu Ser Lys ThrLeu Val 65 70 75 80 His Phe Tyr Pro Met Ala Gly Arg Met Ile Asp Asn IleLeu Val Asp 85 90 95 Cys His Asp Gln Gly Ile Asn Phe Tyr Lys Val Lys IleArg Gly Lys 100 105 110 Met Cys Glu Phe Met Ser Gln Pro Asp Val Pro LeuSer Gln Leu Leu 115 120 125 Pro Ser Glu Val Val Ser Ala Ser Val Pro LysGlu Ala Leu Val Ile 130 135 140 Val Gln Val Asn Met Phe Asp Cys Gly GlyThr Ala Ile Cys Ser Ser 145 150 155 160 Val Ser His Lys Ile Ala Asp AlaAla Thr Met Ser Thr Phe Ile Arg 165 170 175 Ser Trp Ala Ser Thr Thr LysThr Ser Arg Ser Gly Gly Ser Thr Ala 180 185 190 Ala Val Thr Asp Gln LysLeu Ile Pro Ser Phe Asp Ser Ala Ser Leu 195 200 205 Phe Pro Pro Ser GluArg Leu Thr Ser Pro Ser Gly Met Ser Glu Ile 210 215 220 Pro Phe Ser SerThr Pro Glu Asp Thr Glu Asp Asp Lys Thr Val Ser 225 230 235 240 Lys ArgPhe Val Phe Asp Phe Ala Lys Ile Thr Ser Val Arg Glu Lys 245 250 255 LeuGln Val Leu Met His Asp Asn Tyr Lys Ser Arg Arg Gln Thr Arg 260 265 270Val Glu Val Val Thr Ser Leu Ile Trp Lys Ser Val Met Lys Ser Thr 275 280285 Pro Ala Gly Phe Leu Pro Val Val His His Ala Val Asn Leu Arg Lys 290295 300 Lys Met Asp Pro Pro Leu Gln Asp Val Ser Phe Gly Asn Leu Ser Val305 310 315 320 Thr Val Ser Ala Phe Leu Pro Ala Thr Thr Thr Thr Thr ThrAsn Ala 325 330 335 Val Asn Lys Thr Ile Asn Ser Thr Ser Ser Glu Ser GlnVal Val Leu 340 345 350 His Glu Leu His Asp Phe Ile Ala Gln Met Arg SerGlu Ile Asp Lys 355 360 365 Val Lys Gly Asp Lys Gly Ser Leu Glu Lys ValIle Gln Asn Phe Ala 370 375 380 Ser Gly His Asp Ala Ser Ile Lys Lys IleAsn Asp Val Glu Val Ile 385 390 395 400 Asn Phe Trp Ile Ser Ser Trp CysArg Met Gly Leu Tyr Glu Ile Asp 405 410 415 Phe Gly Trp Gly Lys Pro IleTrp Val Thr Val Asp Pro Asn Ile Lys 420 425 430 Pro Asn Lys Asn Cys PhePhe Met Asn Asp Thr Lys Cys Gly Glu Gly 435 440 445 Ile Glu Val Trp AlaSer Phe Leu Glu Asp Asp Met Ala Lys Phe Glu 450 455 460 Leu His Leu SerGlu Ile Leu Glu Leu Ile 465 470 8 439 PRT C. roseus 8 Met Glu Ser GlyLys Ile Ser Val Glu Thr Glu Thr Leu Ser Lys Thr 1 5 10 15 Leu Ile LysPro Ser Ser Pro Thr Pro Gln Ser Leu Ser Arg Tyr Asn 20 25 30 Leu Ser TyrAsn Asp Gln Asn Ile Tyr Gln Thr Cys Val Ser Val Gly 35 40 45 Phe Phe TyrGlu Asn Pro Asp Gly Ile Glu Ile Ser Thr Ile Arg Glu 50 55 60 Gln Leu GlnAsn Ser Leu Ser Lys Thr Leu Val Ser Tyr Tyr Pro Phe 65 70 75 80 Ala GlyLys Val Val Lys Asn Asp Tyr Ile His Cys Asn Asp Asp Gly 85 90 95 Ile GluPhe Val Glu Val Arg Ile Arg Cys Arg Met Asn Asp Ile Leu 100 105 110 LysTyr Glu Leu Arg Ser Tyr Ala Arg Asp Leu Val Leu Pro Lys Arg 115 120 125Val Thr Val Gly Ser Glu Asp Thr Thr Ala Ile Val Gln Leu Ser His 130 135140 Phe Asp Cys Gly Gly Leu Ala Val Ala Phe Gly Ile Ser His Lys Val 145150 155 160 Ala Asp Gly Gly Thr Ile Ala Ser Phe Met Lys Asp Trp Ala AlaSer 165 170 175 Ala Cys Tyr Leu Ser Ser Ser His His Val Pro Thr Pro LeuLeu Val 180 185 190 Ser Asp Ser Ile Phe Pro Arg Gln Asp Asn Ile Ile CysGlu Gln Phe 195 200 205 Pro Thr Ser Lys Asn Cys Val Glu Lys Thr Phe IlePhe Pro Pro Glu 210 215 220 Ala Ile Glu Lys Leu Lys Ser Lys Ala Val GluPhe Gly Ile Glu Lys 225 230 235 240 Pro Thr Arg Val Glu Val Leu Thr AlaPhe Leu Ser Arg Cys Ala Thr 245 250 255 Val Ala Gly Lys Ser Ala Ala LysAsn Asn Asn Cys Gly Gln Ser Leu 260 265 270 Pro Phe Pro Val Leu Gln AlaIle Asn Leu Arg Pro Ile Leu Glu Leu 275 280 285 Pro Gln Asn Ser Val GlyAsn Leu Val Ser Ile Tyr Phe Ser Arg Thr 290 295 300 Ile Lys Glu Asn AspTyr Leu Asn Glu Lys Glu Tyr Thr Lys Leu Val 305 310 315 320 Ile Asn GluLeu Arg Lys Glu Lys Gln Lys Ile Lys Asn Leu Ser Arg 325 330 335 Glu LysLeu Thr Tyr Val Ala Gln Met Glu Glu Phe Val Lys Ser Leu 340 345 350 LysGlu Phe Asp Ile Ser Asn Phe Leu Asp Ile Asp Ala Tyr Leu Ser 355 360 365Asp Ser Trp Cys Arg Phe Pro Phe Tyr Asp Val Asp Phe Gly Trp Gly 370 375380 Lys Pro Ile Trp Val Cys Leu Phe Gln Pro Tyr Ile Lys Asn Cys Val 385390 395 400 Val Met Met Asp Tyr Pro Phe Gly Asp Asp Tyr Gly Ile Glu AlaIle 405 410 415 Val Ser Phe Glu Gln Glu Lys Met Ser Ala Phe Glu Lys AsnGlu Gln 420 425 430 Leu Leu Gln Phe Val Ser Asn 435 9 433 PRT Clarkiabreweri 9 Met Asn Val Thr Met His Ser Lys Lys Leu Leu Lys Pro Ser IlePro 1 5 10 15 Thr Pro Asn His Leu Gln Lys Leu Asn Leu Ser Leu Leu AspGln Ile 20 25 30 Gln Ile Pro Phe Tyr Val Gly Leu Ile Phe His Tyr Glu ThrLeu Ser 35 40 45 Asp Asn Ser Asp Ile Thr Leu Ser Lys Leu Glu Ser Ser LeuSer Glu 50 55 60 Thr Leu Thr Leu Tyr Tyr His Val Ala Gly Arg Tyr Asn GlyThr Asp 65 70 75 80 Cys Val Ile Glu Cys Asn Asp Gln Gly Ile Gly Tyr ValGlu Thr Ala 85 90 95 Phe Asp Val Glu Leu His Gln Phe Leu Leu Gly Glu GluSer Asn Asn 100 105 110 Leu Asp Leu Leu Val Gly Leu Ser Gly Phe Leu SerGlu Thr Glu Thr 115 120 125 Pro Pro Leu Ala Ala Ile Gln Leu Asn Met PheLys Cys Gly Gly Leu 130 135 140 Val Ile Gly Ala Gln Phe Asn His Ile IleGly Asp Met Phe Thr Met 145 150 155 160 Ser Thr Phe Met Asn Ser Trp AlaLys Ala Cys Arg Val Gly Ile Lys 165 170 175 Glu Val Ala His Pro Thr PheGly Leu Ala Pro Leu Met Pro Ser Ala 180 185 190 Lys Val Leu Asn Ile ProPro Pro Pro Ser Phe Glu Gly Val Lys Phe 195 200 205 Val Ser Lys Arg PheVal Phe Asn Glu Asn Ala Ile Thr Arg Leu Arg 210 215 220 Lys Glu Ala ThrGlu Glu Asp Gly Asp Gly Asp Asp Asp Gln Lys Lys 225 230 235 240 Lys ArgPro Ser Arg Val Asp Leu Val Thr Ala Phe Leu Ser Lys Ser 245 250 255 LeuIle Glu Met Asp Cys Ala Lys Lys Glu Gln Thr Lys Ser Arg Pro 260 265 270Ser Leu Met Val His Met Met Asn Leu Arg Lys Arg Thr Lys Leu Ala 275 280285 Leu Glu Asn Asp Val Ser Gly Asn Phe Phe Ile Val Val Asn Ala Glu 290295 300 Ser Lys Ile Thr Val Ala Pro Lys Ile Thr Asp Leu Thr Glu Ser Leu305 310 315 320 Gly Ser Ala Cys Gly Glu Ile Ile Ser Glu Val Ala Lys ValAsp Asp 325 330 335 Ala Glu Val Val Ser Ser Met Val Leu Asn Ser Val ArgGlu Phe Tyr 340 345 350 Tyr Glu Trp Gly Lys Gly Glu Lys Asn Val Phe LeuTyr Thr Ser Trp 355 360 365 Cys Arg Phe Pro Leu Tyr Glu Val Asp Phe GlyTrp Gly Ile Pro Ser 370 375 380 Leu Val Asp Thr Thr Ala Val Pro Phe GlyLeu Ile Val Leu Met Asp 385 390 395 400 Glu Ala Pro Ala Gly Asp Gly IleAla Val Arg Ala Cys Leu Ser Glu 405 410 415 His Asp Met Ile Gln Phe GlnGln His His Gln Leu Leu Ser Tyr Val 420 425 430 Ser 10 445 PRT Dianthuscaryophyllus 10 Met Ser Ile Gln Ile Lys Gln Ser Thr Met Val Arg Pro AlaGlu Glu 1 5 10 15 Thr Pro Asn Lys Ser Leu Trp Leu Ser Asn Ile Asp MetIle Leu Arg 20 25 30 Thr Pro Tyr Ser His Thr Gly Ala Val Leu Ile Tyr LysGln Pro Asp 35 40 45 Asn Asn Glu Asp Asn Ile His Pro Ser Ser Ser Met TyrPhe Asp Ala 50 55 60 Asn Ile Leu Ile Glu Ala Leu Ser Lys Ala Leu Val ProPhe Tyr Pro 65 70 75 80 Met Ala Gly Arg Leu Lys Ile Asn Gly Asp Arg TyrGlu Ile Asp Cys 85 90 95 Asn Ala Glu Gly Ala Leu Phe Val Glu Ala Glu SerSer His Val Leu 100 105 110 Glu Asp Phe Gly Asp Phe Arg Pro Asn Asp GluLeu His Arg Val Met 115 120 125 Val Pro Thr Cys Asp Tyr Ser Lys Gly IleSer Ser Phe Pro Leu Leu 130 135 140 Met Val Gln Leu Thr Arg Phe Arg CysGly Gly Val Ser Ile Gly Phe 145 150 155 160 Ala Gln His His His Val CysAsp Gly Met Ala His Phe Glu Phe Asn 165 170 175 Asn Ser Trp Ala Arg IleAla Lys Gly Leu Leu Pro Ala Leu Glu Pro 180 185 190 Val His Asp Arg TyrLeu His Leu Arg Pro Arg Asn Pro Pro Gln Ile 195 200 205 Lys Tyr Ser HisSer Gln Phe Glu Pro Phe Val Pro Ser Leu Pro Asn 210 215 220 Glu Leu LeuAsp Gly Lys Thr Asn Lys Ser Gln Thr Leu Phe Ile Leu 225 230 235 240 SerArg Glu Gln Ile Asn Thr Leu Lys Gln Lys Leu Asp Leu Ser Asn 245 250 255Asn Thr Thr Arg Leu Ser Thr Tyr Glu Val Val Ala Ala His Val Trp 260 265270 Arg Ser Val Ser Lys Ala Arg Gly Leu Ser Asp His Glu Glu Ile Lys 275280 285 Leu Ile Met Pro Val Asp Gly Arg Ser Arg Ile Asn Asn Pro Ser Leu290 295 300 Pro Lys Gly Tyr Cys Gly Asn Val Val Phe Leu Ala Val Cys ThrAla 305 310 315 320 Thr Val Gly Asp Leu Ser Cys Asn Pro Leu Thr Asp ThrAla Gly Lys 325 330 335 Val Gln Glu Ala Leu Lys Gly Leu Asp Asp Asp TyrLeu Arg Ser Ala 340 345 350 Ile Asp His Thr Glu Ser Lys Pro Gly Leu ProVal Pro Tyr Met Gly 355 360 365 Ser Pro Glu Lys Thr Leu Tyr Pro Asn ValLeu Val Asn Ser Trp Gly 370 375 380 Arg Ile Pro Tyr Gln Ala Met Asp PheGly Trp Gly Ser Pro Thr Phe 385 390 395 400 Phe Gly Ile Ser Asn Ile PheTyr Asp Gly Gln Cys Phe Leu Ile Pro 405 410 415 Ser Arg Asp Gly Asp GlySer Met Thr Leu Ala Ile Asn Leu Phe Ser 420 425 430 Ser His Leu Ser ArgPhe Lys Lys Tyr Phe Tyr Asp Phe 435 440 445 11 440 PRT Taxus cuspidata11 Met Ala Gly Ser Thr Glu Phe Val Val Arg Ser Leu Glu Arg Val Met 1 510 15 Val Ala Pro Ser Gln Pro Ser Pro Lys Ala Phe Leu Gln Leu Ser Thr 2025 30 Leu Asp Asn Leu Pro Gly Val Arg Glu Asn Ile Phe Asn Thr Leu Leu 3540 45 Val Tyr Asn Ala Ser Asp Arg Val Ser Val Asp Pro Ala Lys Val Ile 5055 60 Arg Gln Ala Leu Ser Lys Val Leu Val Tyr Tyr Ser Pro Phe Ala Gly 6570 75 80 Arg Leu Arg Lys Lys Glu Asn Gly Asp Leu Glu Val Glu Cys Thr Gly85 90 95 Glu Gly Ala Leu Phe Val Glu Ala Met Ala Asp Thr Asp Leu Ser Val100 105 110 Leu Gly Asp Leu Asp Asp Tyr Ser Pro Ser Leu Glu Gln Leu LeuPhe 115 120 125 Cys Leu Pro Pro Asp Thr Asp Ile Glu Asp Ile His Pro LeuVal Val 130 135 140 Gln Val Thr Arg Phe Thr Cys Gly Gly Phe Val Val GlyVal Ser Phe 145 150 155 160 Cys His Gly Ile Cys Asp Gly Leu Gly Ala GlyGln Phe Leu Ile Ala 165 170 175 Met Gly Glu Met Ala Arg Gly Glu Ile LysPro Ser Ser Glu Pro Ile 180 185 190 Trp Lys Arg Glu Leu Leu Lys Pro GluAsp Pro Leu Tyr Arg Phe Gln 195 200 205 Tyr Tyr His Phe Gln Leu Ile CysPro Pro Ser Thr Phe Gly Lys Ile 210 215 220 Val Gln Gly Ser Leu Val IleThr Ser Glu Thr Ile Asn Cys Ile Lys 225 230 235 240 Gln Cys Leu Arg GluGlu Ser Lys Glu Phe Cys Ser Ala Phe Glu Val 245 250 255 Val Ser Ala LeuAla Trp Ile Ala Arg Thr Arg Ala Leu Gln Ile Pro 260 265 270 His Ser GluAsn Val Lys Leu Ile Phe Ala Met Asp Met Arg Lys Leu 275 280 285 Phe AsnPro Pro Leu Ser Lys Gly Tyr Tyr Gly Asn Phe Val Gly Thr 290 295 300 ValCys Ala Met Asp Asn Val Lys Asp Leu Leu Ser Gly Ser Leu Leu 305 310 315320 Arg Val Val Arg Ile Ile Lys Lys Ala Lys Val Ser Leu Asn Glu His 325330 335 Phe Thr Ser Thr Ile Val Thr Pro Arg Ser Gly Ser Asp Glu Ser Ile340 345 350 Asn Tyr Glu Asn Ile Val Gly Phe Gly Asp Arg Arg Arg Leu GlyPhe 355 360 365 Asp Glu Val Asp Phe Gly Trp Gly His Ala Asp Asn Val SerLeu Val 370 375 380 Gln His Gly Leu Lys Asp Val Ser Val Val Gln Ser TyrPhe Leu Phe 385 390 395 400 Ile Arg Pro Pro Lys Asn Asn Pro Asp Gly IleLys Ile Leu Ser Phe 405 410 415 Met Pro Pro Ser Ile Val Lys Ser Phe LysPhe Glu Met Glu Thr Met 420 425 430 Thr Asn Lys Tyr Val Thr Lys Pro 435440 12 439 PRT Taxus cuspidata 12 Met Glu Lys Thr Asp Leu His Val AsnLeu Ile Glu Lys Val Met Val 1 5 10 15 Gly Pro Ser Pro Pro Leu Pro LysThr Thr Leu Gln Leu Ser Ser Ile 20 25 30 Asp Asn Leu Pro Gly Val Arg GlySer Ile Phe Asn Ala Leu Leu Ile 35 40 45 Tyr Asn Ala Ser Pro Ser Pro ThrMet Ile Ser Ala Asp Pro Ala Lys 50 55 60 Pro Ile Arg Glu Ala Leu Ala LysIle Leu Val Tyr Tyr Pro Pro Phe 65 70 75 80 Ala Gly Arg Leu Arg Glu ThrGlu Asn Gly Asp Leu Glu Val Glu Cys 85 90 95 Thr Gly Glu Gly Ala Met PheLeu Glu Ala Met Ala Asp Asn Glu Leu 100 105 110 Ser Val Leu Gly Asp PheAsp Asp Ser Asn Pro Ser Phe Gln Gln Leu 115 120 125 Leu Phe Ser Leu ProLeu Asp Thr Asn Phe Lys Asp Leu Ser Leu Leu 130 135 140 Val Val Gln ValThr Arg Phe Thr Cys Gly Gly Phe Val Val Gly Val 145 150 155 160 Ser PheHis His Gly Val Cys Asp Gly Arg Gly Ala Ala Gln Phe Leu 165 170 175 LysGly Leu Ala Glu Met Ala Arg Gly Glu Val Lys Leu Ser Leu Glu 180 185 190Pro Ile Trp Asn Arg Glu Leu Val Lys Leu Asp Asp Pro Lys Tyr Leu 195 200205 Gln Phe Phe His Phe Glu Phe Leu Arg Ala Pro Ser Ile Val Glu Lys 210215 220 Ile Val Gln Thr Tyr Phe Ile Ile Asp Phe Glu Thr Ile Asn Tyr Ile225 230 235 240 Lys Gln Ser Val Met Glu Glu Cys Lys Glu Phe Cys Ser SerPhe Glu 245 250 255 Val Ala Ser Ala Met Thr Trp Ile Ala Arg Thr Arg AlaPhe Gln Ile 260 265 270 Pro Glu Ser Glu Tyr Val Lys Ile Leu Phe Gly MetAsp Met Arg Asn 275 280 285 Ser Phe Asn Pro Pro Leu Pro Ser Gly Tyr TyrGly Asn Ser Ile Gly 290 295 300 Thr Ala Cys Ala Val Asp Asn Val Gln AspLeu Leu Ser Gly Ser Leu 305 310 315 320 Leu Arg Ala Ile Met Ile Ile LysLys Ser Lys Val Ser Leu Asn Asp 325 330 335 Asn Phe Lys Ser Arg Ala ValVal Lys Pro Ser Glu Leu Asp Val Asn 340 345 350 Met Asn His Glu Asn ValVal Ala Phe Ala Asp Trp Ser Arg Leu Gly 355 360 365 Phe Asp Glu Val AspPhe Gly Trp Gly Asn Ala Val Ser Val Ser Pro 370 375 380 Val Gln Gln GlnSer Ala Leu Ala Met Gln Asn Tyr Phe Leu Phe Leu 385 390 395 400 Lys ProSer Lys Asn Lys Pro Asp Gly Ile Lys Ile Leu Met Phe Leu 405 410 415 ProLeu Ser Lys Met Lys Ser Phe Lys Ile Glu Met Glu Ala Met Met 420 425 430Lys Lys Tyr Val Ala Lys Val 435 13 1785 DNA Papaver somniferum CDS(166)..(1587) 13 gagagtttct tcttatccag ctcctcgcaa atgaaatgat tccataatacctctctaaaa 60 gacttggtca ttatataaga gagggagacc acgagcttct tctaaacaacagaaagtatc 120 atctaccatt atcaatcctg ttaaacagtt aaacactttg gatat atg gcaaca atg 177 Met Ala Thr Met 1 tat agt gct gct gtt gaa gtg atc tct aaggaa acc att aaa ccc aca 225 Tyr Ser Ala Ala Val Glu Val Ile Ser Lys GluThr Ile Lys Pro Thr 5 10 15 20 act cca acc cca tct caa ctt aaa aac ttcaat ctg tca ctt ctc gat 273 Thr Pro Thr Pro Ser Gln Leu Lys Asn Phe AsnLeu Ser Leu Leu Asp 25 30 35 caa tgt ttt cct tta tat tat tat gtt cca atcatt ctt ttc tac cca 321 Gln Cys Phe Pro Leu Tyr Tyr Tyr Val Pro Ile IleLeu Phe Tyr Pro 40 45 50 gcc acc gcc gct aat agt acc ggt agc agt aac catcat gat gat ctt 369 Ala Thr Ala Ala Asn Ser Thr Gly Ser Ser Asn His HisAsp Asp Leu 55 60 65 gac ttg ctt aag agt tct ctt tcc aaa aca cta gtt cacttt tat cca 417 Asp Leu Leu Lys Ser Ser Leu Ser Lys Thr Leu Val His PheTyr Pro 70 75 80 atg gct ggt agg atg ata gac aat att ctg gtc gac tgt catgac caa 465 Met Ala Gly Arg Met Ile Asp Asn Ile Leu Val Asp Cys His AspGln 85 90 95 100 ggg att aac ttt tac aaa gtt aaa att aga ggt aaa atg tgtgag ttc 513 Gly Ile Asn Phe Tyr Lys Val Lys Ile Arg Gly Lys Met Cys GluPhe 105 110 115 atg tcg caa ccg gat gtg cca cta agc cag ctt ctt ccc tctgaa gtt 561 Met Ser Gln Pro Asp Val Pro Leu Ser Gln Leu Leu Pro Ser GluVal 120 125 130 gtt tcc gcg agt gtc cct aag gaa gca ctg gtg atc gtt caagtg aac 609 Val Ser Ala Ser Val Pro Lys Glu Ala Leu Val Ile Val Gln ValAsn 135 140 145 atg ttt gac tgt ggt gga aca gcc att tgt tcg agt gta tcacat aag 657 Met Phe Asp Cys Gly Gly Thr Ala Ile Cys Ser Ser Val Ser HisLys 150 155 160 att gcc gat gca gct aca atg agt acg ttc att cgt agt tgggca agc 705 Ile Ala Asp Ala Ala Thr Met Ser Thr Phe Ile Arg Ser Trp AlaSer 165 170 175 180 acc act aaa aca tct cgt agt ggg ggt tca act gct gccgtt aca gat 753 Thr Thr Lys Thr Ser Arg Ser Gly Gly Ser Thr Ala Ala ValThr Asp 185 190 195 cag aaa ttg att cct tct ttc gac tcg gca tct cta ttccca cct agt 801 Gln Lys Leu Ile Pro Ser Phe Asp Ser Ala Ser Leu Phe ProPro Ser 200 205 210 gaa cga ttg aca tct cca tca ggg atg tca gag ata ccattt tcc agt 849 Glu Arg Leu Thr Ser Pro Ser Gly Met Ser Glu Ile Pro PheSer Ser 215 220 225 acc cca gag gat aca gaa gat gat aaa act gtc agc aagaga ttt gtg 897 Thr Pro Glu Asp Thr Glu Asp Asp Lys Thr Val Ser Lys ArgPhe Val 230 235 240 ttc gat ttt gca aag ata aca tct gta cgt gaa aag ttgcaa gta ttg 945 Phe Asp Phe Ala Lys Ile Thr Ser Val Arg Glu Lys Leu GlnVal Leu 245 250 255 260 atg cat gat aac tac aaa agc cgc agg caa aca agggtt gag gtg gtt 993 Met His Asp Asn Tyr Lys Ser Arg Arg Gln Thr Arg ValGlu Val Val 265 270 275 act tct cta ata tgg aag tcc gtg atg aaa tcc actcca gcc ggt ttt 1041 Thr Ser Leu Ile Trp Lys Ser Val Met Lys Ser Thr ProAla Gly Phe 280 285 290 tta cca gtg gta cat cat gcc gtg aac ctt aga aagaaa atg gac cca 1089 Leu Pro Val Val His His Ala Val Asn Leu Arg Lys LysMet Asp Pro 295 300 305 cca tta caa gat gtt tca ttc gga aat cta tct gtaact gtt tcg gcg 1137 Pro Leu Gln Asp Val Ser Phe Gly Asn Leu Ser Val ThrVal Ser Ala 310 315 320 ttc tta cca gca aca aca acg aca aca aca aat gcggtc aac aag aca 1185 Phe Leu Pro Ala Thr Thr Thr Thr Thr Thr Asn Ala ValAsn Lys Thr 325 330 335 340 atc aat agt acg agt agt gaa tca caa gtg gtactt cat gag tta cat 1233 Ile Asn Ser Thr Ser Ser Glu Ser Gln Val Val LeuHis Glu Leu His 345 350 355 gat ttt ata gct cag atg agg agt gaa ata gataag gtc aag ggt gat 1281 Asp Phe Ile Ala Gln Met Arg Ser Glu Ile Asp LysVal Lys Gly Asp 360 365 370 aaa ggt agc ttg gag aaa gtc att caa aat tttgct tct ggt cat gat 1329 Lys Gly Ser Leu Glu Lys Val Ile Gln Asn Phe AlaSer Gly His Asp 375 380 385 gct tca ata aag aaa atc aat gat gtt gaa gtgata aac ttt tgg ata 1377 Ala Ser Ile Lys Lys Ile Asn Asp Val Glu Val IleAsn Phe Trp Ile 390 395 400 agt agc tgg tgc agg atg gga tta tac gag attgat ttt ggt tgg gga 1425 Ser Ser Trp Cys Arg Met Gly Leu Tyr Glu Ile AspPhe Gly Trp Gly 405 410 415 420 aag cca att tgg gta aca gtt gat cca aatatc aag ccg aac aag aat 1473 Lys Pro Ile Trp Val Thr Val Asp Pro Asn IleLys Pro Asn Lys Asn 425 430 435 tgt ttt ttc atg aat gat acg aaa tgt ggtgaa gga ata gaa gtt tgg 1521 Cys Phe Phe Met Asn Asp Thr Lys Cys Gly GluGly Ile Glu Val Trp 440 445 450 gcg agc ttt ctt gag gat gat atg gct aagttc gag ctt cac cta agt 1569 Ala Ser Phe Leu Glu Asp Asp Met Ala Lys PheGlu Leu His Leu Ser 455 460 465 gaa atc ctt gaa ttg att tgatattgcattatctacat gtgttccgta 1617 Glu Ile Leu Glu Leu Ile 470 atcatgattttctccatttc cctttccgta gttggttaca aagaaccaaa taaaggaaaa 1677 gaaaaaacttgtactgctcg atgctttgac attttccatg ttcatccgta aattcccatc 1737 agaaaagagtttcaaatatt agggtattaa aaaaaaaaaa aaaaaaaa 1785 14 474 PRT Papaversomniferum 14 Met Ala Thr Met Tyr Ser Ala Ala Val Glu Val Ile Ser LysGlu Thr 1 5 10 15 Ile Lys Pro Thr Thr Pro Thr Pro Ser Gln Leu Lys AsnPhe Asn Leu 20 25 30 Ser Leu Leu Asp Gln Cys Phe Pro Leu Tyr Tyr Tyr ValPro Ile Ile 35 40 45 Leu Phe Tyr Pro Ala Thr Ala Ala Asn Ser Thr Gly SerSer Asn His 50 55 60 His Asp Asp Leu Asp Leu Leu Lys Ser Ser Leu Ser LysThr Leu Val 65 70 75 80 His Phe Tyr Pro Met Ala Gly Arg Met Ile Asp AsnIle Leu Val Asp 85 90 95 Cys His Asp Gln Gly Ile Asn Phe Tyr Lys Val LysIle Arg Gly Lys 100 105 110 Met Cys Glu Phe Met Ser Gln Pro Asp Val ProLeu Ser Gln Leu Leu 115 120 125 Pro Ser Glu Val Val Ser Ala Ser Val ProLys Glu Ala Leu Val Ile 130 135 140 Val Gln Val Asn Met Phe Asp Cys GlyGly Thr Ala Ile Cys Ser Ser 145 150 155 160 Val Ser His Lys Ile Ala AspAla Ala Thr Met Ser Thr Phe Ile Arg 165 170 175 Ser Trp Ala Ser Thr ThrLys Thr Ser Arg Ser Gly Gly Ser Thr Ala 180 185 190 Ala Val Thr Asp GlnLys Leu Ile Pro Ser Phe Asp Ser Ala Ser Leu 195 200 205 Phe Pro Pro SerGlu Arg Leu Thr Ser Pro Ser Gly Met Ser Glu Ile 210 215 220 Pro Phe SerSer Thr Pro Glu Asp Thr Glu Asp Asp Lys Thr Val Ser 225 230 235 240 LysArg Phe Val Phe Asp Phe Ala Lys Ile Thr Ser Val Arg Glu Lys 245 250 255Leu Gln Val Leu Met His Asp Asn Tyr Lys Ser Arg Arg Gln Thr Arg 260 265270 Val Glu Val Val Thr Ser Leu Ile Trp Lys Ser Val Met Lys Ser Thr 275280 285 Pro Ala Gly Phe Leu Pro Val Val His His Ala Val Asn Leu Arg Lys290 295 300 Lys Met Asp Pro Pro Leu Gln Asp Val Ser Phe Gly Asn Leu SerVal 305 310 315 320 Thr Val Ser Ala Phe Leu Pro Ala Thr Thr Thr Thr ThrThr Asn Ala 325 330 335 Val Asn Lys Thr Ile Asn Ser Thr Ser Ser Glu SerGln Val Val Leu 340 345 350 His Glu Leu His Asp Phe Ile Ala Gln Met ArgSer Glu Ile Asp Lys 355 360 365 Val Lys Gly Asp Lys Gly Ser Leu Glu LysVal Ile Gln Asn Phe Ala 370 375 380 Ser Gly His Asp Ala Ser Ile Lys LysIle Asn Asp Val Glu Val Ile 385 390 395 400 Asn Phe Trp Ile Ser Ser TrpCys Arg Met Gly Leu Tyr Glu Ile Asp 405 410 415 Phe Gly Trp Gly Lys ProIle Trp Val Thr Val Asp Pro Asn Ile Lys 420 425 430 Pro Asn Lys Asn CysPhe Phe Met Asn Asp Thr Lys Cys Gly Glu Gly 435 440 445 Ile Glu Val TrpAla Ser Phe Leu Glu Asp Asp Met Ala Lys Phe Glu 450 455 460 Leu His LeuSer Glu Ile Leu Glu Leu Ile 465 470 15 1425 DNA Papaver somniferum CDS(1)..(1422) 15 atg gca aca atg tat agt gct gct gtt gaa gtg atc tct aaggaa acc 48 Met Ala Thr Met Tyr Ser Ala Ala Val Glu Val Ile Ser Lys GluThr 1 5 10 15 att aaa ccc aca act cca acc cca tct caa ctt aaa aac ttcaat ctg 96 Ile Lys Pro Thr Thr Pro Thr Pro Ser Gln Leu Lys Asn Phe AsnLeu 20 25 30 tca ctt ctc gat caa tgt ttt cct tta tat tat tat gtt cca atcatt 144 Ser Leu Leu Asp Gln Cys Phe Pro Leu Tyr Tyr Tyr Val Pro Ile Ile35 40 45 ctt ttc tac cca gcc acc gcc gct aat agt acc ggt agc agt aac cat192 Leu Phe Tyr Pro Ala Thr Ala Ala Asn Ser Thr Gly Ser Ser Asn His 5055 60 cat gat gat ctt gac ttg ctt aag agt tct ctt tcc aaa aca cta gtt240 His Asp Asp Leu Asp Leu Leu Lys Ser Ser Leu Ser Lys Thr Leu Val 6570 75 80 cac ttt tat cca atg gct ggt agg atg ata gac aat att ctg gtc gac288 His Phe Tyr Pro Met Ala Gly Arg Met Ile Asp Asn Ile Leu Val Asp 8590 95 tgt cat gac caa ggg att aac ttt tac aaa gtt aaa att aga ggt aaa336 Cys His Asp Gln Gly Ile Asn Phe Tyr Lys Val Lys Ile Arg Gly Lys 100105 110 atg tgt gag ttc atg tcg caa ccg gat gtg cca cta agc cag ctt ctt384 Met Cys Glu Phe Met Ser Gln Pro Asp Val Pro Leu Ser Gln Leu Leu 115120 125 ccc tct gaa gtt gtt tcc gcg agt gtc cct aag gaa gca ctg gtg atc432 Pro Ser Glu Val Val Ser Ala Ser Val Pro Lys Glu Ala Leu Val Ile 130135 140 gtt caa gtg aac atg ttt gac tgt ggt gga aca gcc att tgt tcg agt480 Val Gln Val Asn Met Phe Asp Cys Gly Gly Thr Ala Ile Cys Ser Ser 145150 155 160 gta tca cat aag att gcc gat gca gct aca atg agt acg ttc attcgt 528 Val Ser His Lys Ile Ala Asp Ala Ala Thr Met Ser Thr Phe Ile Arg165 170 175 agt tgg gca agc acc act aaa aca tct cgt agt ggg ggt tca actgct 576 Ser Trp Ala Ser Thr Thr Lys Thr Ser Arg Ser Gly Gly Ser Thr Ala180 185 190 gcc gtt aca gat cag aaa ttg att cct tct ttc gac tcg gca tctcta 624 Ala Val Thr Asp Gln Lys Leu Ile Pro Ser Phe Asp Ser Ala Ser Leu195 200 205 ttc cca cct agt gaa cga ttg aca tct cca tca ggg atg tca gagata 672 Phe Pro Pro Ser Glu Arg Leu Thr Ser Pro Ser Gly Met Ser Glu Ile210 215 220 cca ttt tcc agt acc cca gag gat aca gaa gat gat aaa act gtcagc 720 Pro Phe Ser Ser Thr Pro Glu Asp Thr Glu Asp Asp Lys Thr Val Ser225 230 235 240 aag aga ttt gtg ttc gat ttt gca aag ata aca tct gta cgtgaa aag 768 Lys Arg Phe Val Phe Asp Phe Ala Lys Ile Thr Ser Val Arg GluLys 245 250 255 ttg caa gta ttg atg cat gat aac tac aaa agc cgc agg caaaca agg 816 Leu Gln Val Leu Met His Asp Asn Tyr Lys Ser Arg Arg Gln ThrArg 260 265 270 gtt gag gtg gtt act tct cta ata tgg aag tcc gtg atg aaatcc act 864 Val Glu Val Val Thr Ser Leu Ile Trp Lys Ser Val Met Lys SerThr 275 280 285 cca gcc ggt ttt tta cca gtg gta cat cat gcc gtg aac cttaga aag 912 Pro Ala Gly Phe Leu Pro Val Val His His Ala Val Asn Leu ArgLys 290 295 300 aaa atg gac cca cca tta caa gat gtt tca ttc gga aat ctatct gta 960 Lys Met Asp Pro Pro Leu Gln Asp Val Ser Phe Gly Asn Leu SerVal 305 310 315 320 act gtt tcg gcg ttc tta cca gca aca aca acg aca acaaca aat gcg 1008 Thr Val Ser Ala Phe Leu Pro Ala Thr Thr Thr Thr Thr ThrAsn Ala 325 330 335 gtc aac aag aca atc aat agt acg agt agt gaa tca caagtg gta ctt 1056 Val Asn Lys Thr Ile Asn Ser Thr Ser Ser Glu Ser Gln ValVal Leu 340 345 350 cat gag tta cat gat ttt ata gct cag atg agg agt gaaata gat aag 1104 His Glu Leu His Asp Phe Ile Ala Gln Met Arg Ser Glu IleAsp Lys 355 360 365 gtc aag ggt gat aaa ggt agc ttg gag aaa gtc att caaaat ttt gct 1152 Val Lys Gly Asp Lys Gly Ser Leu Glu Lys Val Ile Gln AsnPhe Ala 370 375 380 tct ggt cat gat gct tca ata aag aaa atc aat gat gttgaa gtg ata 1200 Ser Gly His Asp Ala Ser Ile Lys Lys Ile Asn Asp Val GluVal Ile 385 390 395 400 aac ttt tgg ata agt agc tgg tgc agg atg gga ttatac gag att gat 1248 Asn Phe Trp Ile Ser Ser Trp Cys Arg Met Gly Leu TyrGlu Ile Asp 405 410 415 ttt ggt tgg gga aag cca att tgg gta aca gtt gatcca aat atc aag 1296 Phe Gly Trp Gly Lys Pro Ile Trp Val Thr Val Asp ProAsn Ile Lys 420 425 430 ccg aac aag aat tgt ttt ttc atg aat gat acg aaatgt ggt gaa gga 1344 Pro Asn Lys Asn Cys Phe Phe Met Asn Asp Thr Lys CysGly Glu Gly 435 440 445 ata gaa gtt tgg gcg agc ttt ctt gag gat gat atggct aag ttc gag 1392 Ile Glu Val Trp Ala Ser Phe Leu Glu Asp Asp Met AlaLys Phe Glu 450 455 460 ctt cac cta agt gaa atc ctt gaa ttg att tga 1425Leu His Leu Ser Glu Ile Leu Glu Leu Ile 465 470 16 474 PRT Papaversomniferum 16 Met Ala Thr Met Tyr Ser Ala Ala Val Glu Val Ile Ser LysGlu Thr 1 5 10 15 Ile Lys Pro Thr Thr Pro Thr Pro Ser Gln Leu Lys AsnPhe Asn Leu 20 25 30 Ser Leu Leu Asp Gln Cys Phe Pro Leu Tyr Tyr Tyr ValPro Ile Ile 35 40 45 Leu Phe Tyr Pro Ala Thr Ala Ala Asn Ser Thr Gly SerSer Asn His 50 55 60 His Asp Asp Leu Asp Leu Leu Lys Ser Ser Leu Ser LysThr Leu Val 65 70 75 80 His Phe Tyr Pro Met Ala Gly Arg Met Ile Asp AsnIle Leu Val Asp 85 90 95 Cys His Asp Gln Gly Ile Asn Phe Tyr Lys Val LysIle Arg Gly Lys 100 105 110 Met Cys Glu Phe Met Ser Gln Pro Asp Val ProLeu Ser Gln Leu Leu 115 120 125 Pro Ser Glu Val Val Ser Ala Ser Val ProLys Glu Ala Leu Val Ile 130 135 140 Val Gln Val Asn Met Phe Asp Cys GlyGly Thr Ala Ile Cys Ser Ser 145 150 155 160 Val Ser His Lys Ile Ala AspAla Ala Thr Met Ser Thr Phe Ile Arg 165 170 175 Ser Trp Ala Ser Thr ThrLys Thr Ser Arg Ser Gly Gly Ser Thr Ala 180 185 190 Ala Val Thr Asp GlnLys Leu Ile Pro Ser Phe Asp Ser Ala Ser Leu 195 200 205 Phe Pro Pro SerGlu Arg Leu Thr Ser Pro Ser Gly Met Ser Glu Ile 210 215 220 Pro Phe SerSer Thr Pro Glu Asp Thr Glu Asp Asp Lys Thr Val Ser 225 230 235 240 LysArg Phe Val Phe Asp Phe Ala Lys Ile Thr Ser Val Arg Glu Lys 245 250 255Leu Gln Val Leu Met His Asp Asn Tyr Lys Ser Arg Arg Gln Thr Arg 260 265270 Val Glu Val Val Thr Ser Leu Ile Trp Lys Ser Val Met Lys Ser Thr 275280 285 Pro Ala Gly Phe Leu Pro Val Val His His Ala Val Asn Leu Arg Lys290 295 300 Lys Met Asp Pro Pro Leu Gln Asp Val Ser Phe Gly Asn Leu SerVal 305 310 315 320 Thr Val Ser Ala Phe Leu Pro Ala Thr Thr Thr Thr ThrThr Asn Ala 325 330 335 Val Asn Lys Thr Ile Asn Ser Thr Ser Ser Glu SerGln Val Val Leu 340 345 350 His Glu Leu His Asp Phe Ile Ala Gln Met ArgSer Glu Ile Asp Lys 355 360 365 Val Lys Gly Asp Lys Gly Ser Leu Glu LysVal Ile Gln Asn Phe Ala 370 375 380 Ser Gly His Asp Ala Ser Ile Lys LysIle Asn Asp Val Glu Val Ile 385 390 395 400 Asn Phe Trp Ile Ser Ser TrpCys Arg Met Gly Leu Tyr Glu Ile Asp 405 410 415 Phe Gly Trp Gly Lys ProIle Trp Val Thr Val Asp Pro Asn Ile Lys 420 425 430 Pro Asn Lys Asn CysPhe Phe Met Asn Asp Thr Lys Cys Gly Glu Gly 435 440 445 Ile Glu Val TrpAla Ser Phe Leu Glu Asp Asp Met Ala Lys Phe Glu 450 455 460 Leu His LeuSer Glu Ile Leu Glu Leu Ile 465 470 17 1514 DNA Papaver somniferum 17ccattatcaa tcctgttaaa cagttaaaca ctttggatat atggcaacaa tgtatagtgc 60tgctgttgaa gtgatctcta aggaaaccat taaacccaca actccaaccc catctcaact 120taaaaacttc aatctgtcac ttctcgatca atgttttcct ttatattatt atgttccaat 180cattcttttc tacccagcca ccgccgctaa tagtaccggt agcagtaacc atcatgatga 240tcttgacttg cttaagagtt ctctttccaa aacactagtt cacttttatc caatggctgg 300taggatgata gacaatattc tggtcgactg tcatgaccaa gggattaact tttacaaagt 360taaaattaga ggtaaaatgt gtgacttcat gtcgcaaccg gatgtgccac taagccagct 420tcttccctct gaaattgttt ccgcgagtgt ccctaaggaa gcactggtga tcgttcaagt 480gaacatgttt gactgtggtg gaacagccat ttgttcgagt gtatcacata agattgcgga 540tgcagctaca atgagtacgt tcattcgtag ttgggcaagc accactaaaa catctcgtag 600tgggggtgca actgctgccg ttacagatca gaaattgatt ccttctttcg actcggcatc 660tctattccca cctagtgaac gattgacatc tccatcaggg atgtcagaga taccattttc 720cagtacccca gaggatacag aagatgataa aactgtcagc aagagatctg tgttcgattt 780tgcaaagata acatctgtac gtgaaaagtt gcaagtattg atgcatgata actacaaaag 840ccgcaggcca acaagggttg aggtggttac ttctctaata tggaagtccg tgatgaaatc 900cactccagcc ggttttttac cagtggtaca tcatgccgtg aaccttagaa agaaaatgga 960cccaccatta caagatgttt cattcggaaa tctatctgta actgtttcgg cgttcttacc 1020agcaacaaca acgacaacaa caaatgcggt caacaagaca atcaatagta cgagtagtga 1080atcacaagtg gtacttcatg agttacatga ttttatagct cagatgagga gtgaaataga 1140taaggtcaag ggtgataaag gtagcttgga gaaagtcatt caaaattttg cttctggtca 1200tgatgcttca ataaagaaaa tcaatgatgt tgaagtgata aacttttgga taagtagctg 1260gtgcaggatg ggattatacg agattgattt tggttgggga aagccaattt gggtaacagt 1320tgatccaaat atcaagccga acaagaattg ttttttcatg aatgatacga aatgtggtga 1380aggaatagaa gtttgggcga gctttcttga ggatgatatg gctaagttcg agcttcacct 1440aagtgaaatc cttgaattga tttgatattg cattatctac atgtgttccg taatcatgat 1500tttctccatt tccc 1514 18 38 DNA Artificial Sequence Description ofArtificial Sequence Avr II primer 18 ccctagggga aatggagaaa atcatgattacggaacac 38 19 38 DNA Artificial Sequence Description of ArtificialSequence Xho I primer 19 cctcgagcca ttatcaatcc tgttaaacag ttaaacac 38

What is claimed is:
 1. A protein comprising consectutive amino acids,the amino acid sequence of which i) is the amino acid sequenceillustrated in FIG. 8 (SEQ. ID No. 14) or, ii) a fragment of the aminoacid sequence illustrated in FIG. 8 (SEQ. ID No. 14), said fragmenthaving at least 15 amino acids, or iii) a variant of the amino acidsequence illustrated in FIG. 8 (SEQ. ID No. 14), said variant having atleast 70% identity with the amino acid sequence of FIG. 8 (SEQ. ID No.14) over a length of at least 400 amino acids.
 2. The protein accordingto claim 1 having salutaridinol 7-O-acetyltransferase activity.
 3. Theprotein according to claim 1 or 2, the sequence of which is a fragmentof the amino acid sequence illustrated in FIG. 8 (SEQ. ID No. 14), saidfragment having a length of 15 to 470 amino acids.
 4. The proteinaccording to claim 3, wherein the fragment has a length of 20 to 350amino acids.
 5. The protein according to claim 1 or 2 which is a variantof the amino acid sequence of which is illustrated in FIG. 8 (SEQ. IDNo. 14), wherein said variant has at least 70% identity with the aminoacid sequence of FIG. 8 (SEQ. ID No. 14) over a length of at least 400amino acids, and differs therefrom by insertion, replacement and/ordeletion of at least one amino acid.
 6. The protein according to claim 5comprising the protein encoded by the nucleic acid sequence SAT 2 CO48illustrated in FIG. 11 (SEQ. ID No. 17).
 7. A peptide comprising afragment of salutaridinol 7-O-acetyltransferase protein, said fragmentcomprising at least 6 consecutive amino acids which is not present inother plant acetyltransferases as illustrated in FIG. 2 (SEQ. ID Nos. 8to 12).
 8. The peptide according to claim 7 wherein said fragment has alength of 10 to 20 amino acids.
 9. A nucleic acid encoding a proteinaccording to any one of claims 1 to
 8. 10. Nucleic acid, comprisingconsecutive nucleotides, the nucleotide sequence of which i) is thenucleic acid sequence illustrated in FIG. 9 (SEQ. ID No. 13) or FIG. 10(SEQ. ID No. 15), or ii) a fragment of the nucleic acid sequenceillustrated in FIG. 9 (SEQ. ID No. 13) or FIG. 10 (SEQ. ID No. 15), saidfragment having a length of at least 45 nucleotides, or iii) a variantof the sequence illustrated in FIG. 9 (SEQ. ID No. 13) or FIG. 10 (SEQ.ID No. 15), said variant having at least 70% identity with the sequenceof FIG. 9 (SEQ. ID No. 13) or FIG. 10 (SEQ. ID No. 15) over a length ofat least 1200 bases, or iv) a sequence complementary to sequences (i),(ii) or (iii), or v) the RNA equivalent of any of sequences (i), (ii),(iii) or (iv).
 11. The nucleic acid according to claim 10 encoding aprotein having salutaridinol 7-O-acetyltransferase activity.
 12. Thenucleic acid according to claim 10 or 11, the sequence of whichcomprises a fragment of the sequence illustrated in FIG. 9 (SEQ. ID No.13) or FIG. 10 (SEQ. ID No. 15), said fragment having a length of 45 to1420 nucleotides.
 13. The nucleic acid according to claim 12, whereinthe fragment has a length of 50 to 1000 nucleotides.
 14. The nucleicacid according to claim 10 or 11, the sequence of which is a variant ofthe nucleotide sequence illustrated in FIG. 10 (SEQ. ID No. 15), whereinsaid variant has at least 70% identity with the sequence of FIG. 10(SEQ. ID No. 15) over a length of at least 1200 bases and differstherefrom by insertion, replacement and/or deletion of at least onenucleotide.
 15. The nucleic acid according to claim 14, the sequence ofwhich comprises the coding sequence of the molecule SAT 2 CO48illustrated in FIG. 11 (SEQ. ID No. 17).
 16. The nucleic acid accordingto claim 14, wherein the variant has the capacity to hybridise to thesequence illustrated in FIG. 10 (SEQ. ID No. 15) under stringenthybridization conditions.
 17. The nucleic acid comprising a fragment ofa salutaridinol 7-O-acetyltransferase gene, said fragment comprises atleast 18 consecutive nucleotides unique to the salutaridinol7-O-acetyltransferase gene and being chosen from the 5′ or 3′untranslated regions of the sequence illustrated in FIG. 9 (SEQ. ID No.13), or a sequence which is complementary thereto.
 18. The nucleic acidaccording to any one of claims 9 to 17 operably linked to atranscription regulatory sequences.
 19. A chimeric gene comprising anucleotide corresponding to a coding sequence operably linked to anucleotide corresponding toat least one heterologous transcriptionalregulatory sequence, said coding sequence nucleotides being a nucleicacid according to any one of claims 9 to
 17. 20. A cell transformed ortransfected by a nucleic acid according to any one of claims 9 to 18, orby a chimeric gene according to claim
 19. 21. The cell according toclaim 20 which is a prokaryotic cell.
 22. The cell according to claim 21which is a bacterial cell.
 23. That cell according to claim 20 which isa eukaryotic cell.
 24. The cell according to claim 23 which is a yeastcell.
 25. The cell according to claim 23 which is a vertebrate cell oran invertebrate cell.
 26. The cell according to claim 23 which is amammalian cell or an insect cell.
 27. The cell according to claim 23which is a plant cell.
 28. The cell according to claim 27 which is adifferentiated plant cell.
 29. The cell according to claim 27 which is amonocotyledonous or dicotyledonous plant cell.
 30. The cell according toclaim 27 which is a cell of a plant which naturally contains a geneencoding salutaridinol 7-O-acetyltransferase.
 31. The cell according toclaim 30, which is a cell of a plant belonging to the genus Papaver. 32.The cell according to claim 31 wherein the cell is a Papaver somniferum,Papaver bracteatum, Papaver cylindricum, Papaver orientale, Papaversetigerum, Papaver pseudo-orientale, Papaver lauricola, Papaverpersicum, Papaver caucasium, or Papaver carmeli cell.
 33. The cellaccording to claim 27 which is a cell of a plant which does notnaturally contain a gene encoding salutaridinol 7-O-acetyltransferase.34. The cell according to claim 30 which is a cell of a plant belongingto a family chosen from Papaveraceae family, the Euphorbiaceae family,the Berberidaceae family, the Fumariaceae family, or the Ranunculaceaefamily.
 35. The cell according to claim 34 which is a cell of a plant ofa species chosen from Berberis spp., Podophyllum spp., Adlumia spp.,Cordyalis spp., Dicentra spp., Fumaria spp., Papaver spp., Glauciumspp., Argemone spp., Croton spp., Eschscholzia spp., or Bocconia spp.36. A transgenic plant transformed or transfected with a nucleic acidaccording to any one of claims 9 to 18, or comprising a cell accordingto any one of claims 27 to
 35. 37. A transgenic plant according to claim36, wherein said plant belongs to the genus Papaver.
 38. The transgenicplant according to claim 37, wherein said plant belongs to a specieschosen from Papaver somniferum, Papaver bracteatum, Papaver cylindricum,Papaver orientate, Papaver setigerum, Papaver pseudo-orientale, Papaverlauricola, Papaver persicum, Papaver caucasium, or Papaver carmeli. 39.The transgenic plant according to claim 38 which exhibitsover-expression of salutaridinol 7-O-acetyltransferase.
 40. Thetransgenic plant according to any one of claims 36 to 38 which exhibitsreduced expression of salutaridinol 7-O-acetyltransferase.
 41. A seed ofthe transgenic plant according to any one of claims 36 to
 40. 42. Amethod for producing pentacyclic morphinan alkaloids, said methodcomprising the steps of: i) introducing a nucleic acid encodingsalutaridinol 7-O-acetyltransferase into a plant cell which is capableof expressing salutaridinol and/or salutaridine and/or (R)-reticuline,ii) propagating said plant cell to produce a multiplicity ofmorphinan-producing cells, and iii) recovering said morphinan(s) fromsaid multiplicity of cells.
 43. The method according to claim 42,wherein the multiplicity of cells is a cell culture of differentiated orundifferentiated cells.
 44. The method according to claim 42, whereinthe multiplicity of cells is a transgenic plant.
 45. The methodaccording to any one of claims 42 to 44, wherein said plant cell is acell of a plant which naturally has the capacity to produce thebaine.46. The method according to claim 45, wherein said plant cell has thefurther capacity to prodiuce morphine, codeine, and/or oripavine. 47.The method according to claim 45, wherein said cell is a cell of a plantchosen from a member of the Papaveraceae family.
 48. The methodaccording to claim 47, wherein said cell is a cell of a plant chosenfrom a member of the genus Papaver.
 49. The method according to claim48, wherein said plant is chosen from Papaver somniferum, Papaverbracteatum, Papaver cylindricum, Papaver orientate, Papaver setigerum,Papaver pseudo-orientale, Papaver lauricola, Papaver persicum, Papavercaucasium, or Papaver carmeli; and the morphinan(s) produced includethebaine
 50. The method according to claim 47 wherein said plant ischosen from Papaver somniferum, Papaver bracteatum, Papaver cylindricum,Papaver orientale, or Papaver setigerum, and the morphinan(s) producedinclude morphine and/or codeine and/or oripavine
 51. The methodaccording to any one of claims 42 to 44, wherein said plant cell is acell of a plant which does not naturally have the capacity to producethebaine.
 52. The method according to claim 51, wherein said cell is acell of a plant chosen from a member of the Papaveraceae family, theEuphorbiaceae family, the Berberidaceae family, the Fumariaceae family,the Ranunculaceae family.
 53. The method according to claim 52, whereinsaid plant is chosen from Papaver lasiothrix, Papaver nudicaule, Papaveratlanticum, Glaucium spp., Croton spp., or Eschscholzia spp.
 54. Themethod according to claim 53, wherein the morphinan(s) produced includethebaine.
 55. The method according to any one of claims 42 to 54,wherein said nucleic acid molecule is a molecule according to claim 11.56. A method for the production of thebaine, said method comprising thesteps of: i) contacting in vitro a-protein having salutaridinol7-O-acetyltransferase activity with salutaridinol and acetyl co-enzyme Aat pH 8 to 9, and ii) recovering the thebaine thus produced.
 57. Themethod according to claim 56 wherein the protein having salutaridinol7-O-acetyltransferase activity is a protein according to claim
 2. 58. Amethod for producing a plant having altered alkaloid production, saidmethod comprising the steps of: i) transforming or transfecting a cellof an alkaloid-producing plant, or a part of an alkaloid-producing plantwith a nucleic acid according to any one of claims 9 to 18, inconditions which permit the expression of the nucleic acid molecule, andii) optionally regenerating a whole plant from the said cell.
 59. Amethod for producing a plant cell culture having altered alkaloidproduction, said method comprising the steps of: i) transforming ortransfecting a cell of an alkaloid-producing plant with a nucleic acidaccording to any one of claims 9 to 18, in conditions which permit theexpression of the nucleic acid molecule, and ii) establishing a cellculture from said cell.
 60. The method according to claim 58 or 59,wherein the alkaloid-producing plant produces an alkaloid which is afive-ring morphinan alkaloid.
 61. The method according to claim 58 or59, wherein the alkaloid-producing plant produces at least one ofthebaine, oripavine, codeine and morphine.
 62. the method according toclaim 61, wherein the alkaloid-producing plant is a member of the genusPapaver.
 63. The method according to claim 62, wherein thealkaloid-producing plant is Papaver somniferum, Papaver bracteatum,Papaver cylindricum, Papaver orientale, Papaver setigerum, Papaverpseudo-orientale, Papaver lauricola, Papaver persicum, Papavercaucasium, or Papaver carmeli.
 64. The method according to any one ofclaims 52 to 57, wherein the nucleic acid molecule encodes a proteinhaving salutaridinol 7-O-acetyltransferase activity, thereby increasingthe expression level of salutaridinol 7-O-acetyltransferase in theplant.
 65. The method according to any one of claims 58 to 63, whereinthe nucleic acid molecule encodes an inhibitor of salutaridinol7-O-acetyltransferase activity.
 66. The method of claim 65, wherein theinhibitor is a ribozyme or antisense molecule, so as to thereby decreasethe expression level of salutaridinol 7-O-acetyltransferase RNA in theplant.
 67. The method according to any one of claims 58 to 63 whereinthe alteration in alkaloid production comprises an alteration in totalalkaloid yield, or in the type of alkaloid, or in the relativeproportions of different alkaloids, produced by the plant.
 68. A planthaving altered alkaloid production, said plant being obtainable by amethod according to any one of claims 58, 60 to
 66. 69. The plantaccording to claim 68 which is an alkaloid poppy plant.
 70. The plantaccording to claim 69 which is Papaver somniferum, Papaver bracteatum,Papaver cylindricum, Papaver orientate, Papaver setigerum, Papaverpseudo-orientate, Papaver lauricola, Papaver persicum, Papavercaucasium, or Papaver carmeli.
 71. Straw of a plant according to any oneof claims 68 to
 70. 72. Concentrate of straw of a plant according to anyone of claims 68 to
 70. 73. A method for producing a protein havingsalutaridinol 7-O-acetyltransferase activity, said method comprising i)transforming or transfecting a cell with a nucleic acid according toclaim 11, in conditions permitting the expression of the protein havingsalutaridinol 7-O-acetyltransferase activity, ii) propagating saidcells, and iii) recovering the thus-produced protein havingsalutaridinol 7-O-acetyltransferase activity.